Pd-l1 expressing hematopoietic stem cells and uses

ABSTRACT

Embodiments disclosed here provide engineered modified hematopoietic stem cells (HSCs), artificially prostaglandin E2 (PGE2)-stimulated HSCs, compositions comprising these HSCs, methods of using these modified HSCs for treating autoimmune diseases and disorders and for suppressing the immune system. In particular, the engineered modified HSCs or PGE2-stimulated HSCs express the surface marker, programmed cell death-1 ligand 1 (PD-L1).

CROSS REFERENCE TO RELATED APPLICATION

This application is a Continuation application of co-pending U.S. patentapplication Ser. No. 16/673,283, filed Nov. 4, 2019, which is aContinuation application of Ser. No. 16/215,932, filed Dec. 11, 2018,now U.S. Pat. No. 10,517,899, issued Dec. 31, 2019, which is aContinuation application of U.S. patent application Ser. No. 15/745,553,filed Jan. 17, 2018, now abandoned, which is a 35 U.S.C. § 371 NationalPhase Entry application of International Application No.PCT/US2016/043053 filed Jul. 20, 2016, which designates the U.S., andwhich claims benefit under 35 U.S.C. § 119(e) of U.S. provisionalapplication No. 62/194,969 filed Jul. 21, 2015, the contents of each ofwhich are hereby incorporated by reference in their entirety.

SEQUENCE LISTING

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BACKGROUND

Immunological approaches have failed in the treatment of autoimmunediseases thus far. For example, in the long-term treatment of autoimmunetype 1 diabetes (T1D). Despite considerable effort to halt or delay thedestruction of beta-cells in T1D, success remains elusive. Historically,approaches aiming to treat T1D have made a negligible number of subjectsinsulin-independent. The Diabetes Control and Complications Trial (DCCT)have demonstrated that improving glucose control and preserving β-cellfunction in individuals with T1D lowered the incidence of diabeticcomplications.

Stem cells have been used for autoimmune diabetes treatment. Mesenchymalstem cells (MSCs) are fibroblast-like non-hematopoietic progenitor cellswith the capacity for adipogenic, chondrogenic, and osteogenicdifferentiation. MSCs, because of their immunomodulatory properties andtheir potential to differentiate into insulin-producing cells, representa viable therapeutic option for autoimmune diabetes A study showedshort-term reversal of diabetes in 88% of BALB/c-MSC-treatedhyperglycemic NOD mice. However, NOD mice treated with NOD-MSCs remainedhyperglycemic. Further reports indicated that treatment with congenicNOR-MSCs resulted in a more pronounced and prolonged reversal ofhyperglycemia in treated NOD mice (88% and 62% short-term and long-termreversal respectively), suggesting the potential use of haplo-identicalMSCs in autoimmune diabetes. Based on this data, a clinical trial wasinitiated in the US by the JDRF and by the Osiris Corporation, butinterim unpublished results at 1-year of follow-up were disappointing.Furthermore, safety concerns primarily related to potential oncogenictransformation of MSCs may limit their use in the clinical setting.(Moufida Ben Nasr et al., (2015), “The rise, fall, and resurgence ofimmunotherapy in type 1 diabetes. Pharmacological Research”, 98:31-38).

Hematopoietic stem cells (HSCs) transplantation has been reported toyield promising results in long term treatment of T1D. However,accumulating clinical data show limited success for long-term insulinindependence and for a limited population with the condition. HSCs mayprovide treatment solutions because HSCs are endowed withimmunoregulatory properties and can induce central and peripheralimmunological tolerance per se. In 2003, Voltarelli et al. 2007 (JAMA,297:1568-76) initiated a phase I/II study in (T1D), to evaluate thesafety and efficacy of autologous HSC transplantation (AHSCT) using acombined regimen of thymoglobulin plus cyclophosphamide. The latestanalysis reported 20 out of 23 of the treated patients with a meanfollow-up of 30 months, insulin-free for more than 1 year. However, inthe aforementioned studies, it is difficult to distinguish between theeffects of concomitant immunosuppressants and the mechanisms ofHSC-mediated immunomodulation.

A report from a multicenter analysis on 65 newly-diagnosed T1Dindividuals treated with AHSCT using a similar protocol to thatpreviously reported showed that insulin independence in nearly 60% oftreated subjects was achieved. However, several adverse events have beenrecorded suggesting this as a therapy for selected T1D individuals only.

Moreover, the AHSCT protocols used in these studies were designed foradults and not for pediatric subjects with T1D, and thus AHSCT can beonly considered for a well-defined group of individuals that may benefitfrom this treatment.

HSCs are endowed with immunoregulatory properties. Preclinical studiesdemonstrated that T cell-depleted bone marrow-resident CD34+ stem cellsovercome MHC barriers in sublethally irradiated mice and that murineHSCs may delete effector cells. This effect can be reverted by theaddition of a caspase inhibitor, suggesting a deletion-based mechanism.With respect to human HSCs, the human CD34⁺ population have been shownto be endowed with potent veto activity and neutralized precursors ofcytotoxic T lymphocytes (CTLs) directed against their antigens.

Based on that principle, research focused on finding additionalimmunological strategies to prevent β-cell loss in subjects with a newlydiagnosed T1D have been initiated. Since then, the search for feasibleand safe immunological approaches in order to re-establish tolerancetoward islet autoantigens (and preserve β-cell function) is ongoing.

SUMMARY

Embodiments of the present disclosure provide programmed cell death-1ligand 1 (PD-L1) expressing hematopoietic stem cells (HSCs), methods ofmaking these cells, and therapeutic methods of using these cells for thetreatment of autoimmune diseases such as type 1 diabetes (T1D), and forthe suppression of the immune system in a subject. For example, thetherapeutic methods are useful after an organ or bone marrowtransplantation, and when a subject has a defect in producing PD-L1⁺expressing HSCs, e.g. in Type 1 diabetes (T1D). The disclosure providesPD-L1⁺ expressing HSCs that are stimulated by prostaglandin E2 (PGE₂)treatment or by transduction with an exogenous copy of a nucleic acidthat encodes for the PD-L1 protein for promoting PD-L1 expression in thecell after transduction of the nucleic acid.

Type 1 diabetes (T1D) mouse models and human T1D patients have fewerHSCs that express PD-L1 and these HSCs express lower amounts of PD-L1.Supplementing the missing PD-L1 promote immune tolerance prolongsurvival of transplanted islet grafts in mouse model of T1D and in T1Dsubjects.

The present disclosure provides that PGE₂-stimulated HSCs promote immunetolerance and prolong survival of transplanted islet grafts in mousemodel of T1D. The PGE₂-stimulated HSCs are now re-programmed to expressPD-L1 prior to the PGE₂-stimulation. The PGE₂-stimulated HSCs also arenow re-programmed to express more PD-L1 prior to the PGE₂-stimulation.This HSC-mediated immune tolerance occurs via the programmed celldeath-1 (PD-1) pathway. Programmed cell death-1 receptor (PD-1) is foundon activated T-cells; the programmed cell death-1 receptor ligand(PD-L1, also known as B7-H1) is expressed in other cells, e.g. HSC. Thereception/ligand PD-L1/PD-1 interaction deactivates T cell's cytotoxicactivity and leads to the immune system inhibition and tolerance.

Moreover, the present disclosure provides that in vivo administration ofanti-PD-1 mAb, PIM2, in NOD mice delayed the onset of diabetes and alsodelayed the islet allografts rejection. A NOD mouse is the mouse modelof human T1D. If a human is at high risk for developing T1D,administering the PD-L1⁺ cells can delay the onset of T1D too.Furthermore, this disclosure provides that the PD-L1 expression in HSCcan be increased by: (a) an overexpression of a PD-L1 cDNA, e.g., via alentiviral system or an avian virus system or an adeno-associated virussystem; and (b) ex vivo culture of HSC in PGE₂, ie., contact with PGE₂.

Accordingly, in one embodiment, it is the objective of this disclosureto provide modified PD-L1+ expressing HSCs produced by theoverexpression of an exogenous copy of a PD-L1 cDNA in the HSCs. Theexogenous copy of cDNA has been introduced or transfected into the HSCs.

In one embodiment, it is the objective of this disclosure to provide anex vivo method of producing a population of PD-L1⁺ expressing HSCs by acontact or stimulation with PGE₂. The inventors found that under certainconditions, PGE₂ stimulates endogenous expression of PD-L1 in HSCs, eventhe defective HSCs from T1D that have lower expression of PD-L1.

In one embodiment, it is the objective of this disclosure to provide anex vivo method of producing a population of PD-L1+ expressing HSCs bythe overexpression of an exogenous copy of a PD-L1 cDNA.

In one embodiment, it is the objective of this disclosure to provide amethod of treating autoimmune disease or suppressing the immune systemby using the PD-L1⁺ expressing HSCs described here.

Accordingly, in one embodiment, provided herein is a population ofmodified HSCs where the cells carry an exogenous copy of a nucleic acidencoding a PD-L1 or the HSCs are ex vivo stimulated by PGE₂ describedherein to stimulate PD-L1 expression the cells.

In one embodiment, provided herein is a population of modified HSCs foruse in the prevention and treatment of an autoimmune disease or disorderin a subject, for use in suppressing an immune response in a subject,for use in the delay of the onset of T1D in a subject at risk ofdeveloping T1D, for use in preventing or delaying an allogenictissue/organ rejection in a subject, and for use in the treatment of T1Din subjects (adult and pediatric T1D patients). In one embodiment, themodified HSCs carry an exogenous copy of a nucleic acid encoding aPD-L1. The modified HSCs express more PD-L1 compared to non-modifiedcells not carrying an exogenous copy of a nucleic acid encoding a PD-L1.In another embodiment, the modified HSCs have been ex vivo stimulated byPGE₂ via methods described herein to stimulate PD-L1 expression thecells. In one embodiment, there are more PD-L1 expressing cells in thepopulation of cells after PGE₂ stimulation. In another embodiment, thePGE₂ stimulated cells express more PD-L1 after stimulation compared toprior to the stimulation.

In one embodiment, provided herein is a population of modified HSCs foruse in the manufacture of medicament for the prevention and treatment ofan autoimmune disease or disorder in a subject, for the suppressing animmune response in a subject, for delaying of the onset of T1D in asubject at risk of developing T1D, for use in preventing or delaying anallogenic tissue/organ rejection in a subject, and for the treatment ofT1D in subjects (adult and pediatric T1D patients). In one embodiment,the modified HSCs carry an exogenous copy of a nucleic acid encoding aPD-L1. In another embodiment, the modified HSCs have been ex vivostimulated by PGE₂ via methods described herein to stimulate PD-L1expression in the cells.

In one embodiment, provided herein is a composition comprising apopulation of modified HSCs described herein, where the cells carry anexogenous copy of a nucleic acid encoding a PD-L1.

In one embodiment, provided herein is a composition for transplantationinto a subject, for the prevention and treatment of an autoimmunedisease or disorder, for suppressing/reducing an immune response in asubject, for use in the delay of the onset of T1D in a subject at riskof developing T1D, for use in preventing or delaying an allogenictissue/organ rejection in a subject, and for the treatment of T1D inadult and pediatric subjects, the composition comprising the modifiedHSCs described herein, where the HSCs are modified and carry anexogenous copy of a nucleic acid encoding a PD-L1 or the HSCs are exvivo stimulated by PGE₂ via methods described herein to stimulate PD-L1expression in the cells. In some embodiment, the HSCs are ex vivostimulated with both PGE₂ and a steroid such as dexamethasone.

In one embodiment, provided herein is a composition the modified HSCsdescribed herein for the manufacture of medicament for use intransplantation into a subject, for the prevention and treatment of anautoimmune disease or disorder, for suppressing/reducing an immuneresponse in a subject, for use in the delay of the onset of T1D in asubject at risk of developing T1D, for use in preventing or delaying anallogenic tissue/organ rejection in a subject, and for the treatment ofT1D in adult and pediatric subjects, where the HSCs are modified andcarry an exogenous copy of a nucleic acid encoding a PD-L1 or the HSCsare ex vivo stimulated by PGE₂ via methods described herein to stimulatePD-L1 expression in the cells. In some embodiment, the HSCs are ex vivostimulated with both PGE₂ and a steroid such as dexamethasone.

In one embodiment of any one of the population of HSCs or compositioncomprising a population of HSCs, the HSCs are expressing PD-L1. Inanother embodiment, the HSCs exhibit increased PD-L1 expression. In yetanother embodiment, the population of HSCs exhibits an increaseproportion of PD-L1⁺ expressing cells, e.g., an increase of at least onefold.

In one embodiment of any one of the population of HSCs or compositioncomprising a population of HSCs, the nucleic acid is a copy DNA (cDNA).

In one embodiment of any one of the population of HSCs or compositioncomprising a population of HSCs, the nucleic acid is a genomic DNA.

In one embodiment of any one of the population of HSCs or compositioncomprising a population of HSCs, the nucleic acid is integrated into thegenome of the cells.

In one embodiment of any one of the population of HSCs or compositioncomprising a population of HSCs, the nucleic acid is introduced into theHSCs via a vector.

In one embodiment of any one of the population of HSCs or compositioncomprising a population of HSCs, the vector is a viral vector.

In one embodiment of any one of the population of HSCs or compositioncomprising a population of HSCs, the viral vector is a lentiviralvector, an avian virus vector or an adeno-associated virus.

In one embodiment of any one of the population of HSCs or compositioncomprising a population of HSCs, the HSCs are mammalian cells.

In one embodiment of any one of the population of HSCs or compositioncomprising a population of HSCs, the mammalian cells are human cells.

In one embodiment of any one of the population of HSCs or compositioncomprising a population of HSCs, prior to the modification with a vectordescribed herein or stimulation with PGE₂ described, the HSCs areobtained from the bone marrow, umbilical cord, amniotic fluid, chorioniccord blood, placental blood or peripheral blood.

In one embodiment of any one of the population of HSCs or compositioncomprising a population of HSCs, the HSCs are obtained from mobilizedperipheral blood.

In one embodiment of any one of the population of HSCs or compositioncomprising a population of HSCs, the HSCs are derived from a healthyindividual.

In one embodiment of any one of the population of HSCs or compositioncomprising a population of HSCs, the HSCs are derived from an individualwith a diagnosed disease or disorder, or an individual who is an organor bone marrow transplant recipient.

In one embodiment of any one of the population of HSCs or compositioncomprising a population of HSCs, the HSCs are derived from an individualwho has newly been diagnosed with T1D.

In one embodiment of any one of the population of HSCs or compositioncomprising a population of HSCs, the HSCs are derived from an individualwho has newly been detected to have self-autoantibodies associated withT1D, e.g., GAD65 autoantibody, and islet antigen 2 autoantibody.

In one embodiment of any one of the population of HSCs or compositioncomprising a population of HSCs, the diagnosed disease or disorder is anautoimmune disease or disorder.

In one embodiment of any one of the population of HSCs or compositioncomprising a population of HSCs, the autoimmune disease or disorder isT1D.

In one embodiment of any one of the population of HSCs or compositioncomprising a population of HSCs, the cells are ex vivo cultured beforethe introduction of the exogenous copy of a nucleic acid encoding aPD-L1, or after the introduction of the exogenous copy of a nucleic acidencoding a PD-L1, or both before and after the introduction of theexogenous copy of a nucleic acid encoding a PD-L1.

In one embodiment of any one of the population of HSCs or compositioncomprising a population of HSCs, the cells are cryopreserved prior tothe introduction of the exogenous copy of a nucleic acid encoding aPD-L1, or after the introduction of the exogenous copy of a nucleic acidencoding a PD-L1, or both before and after the introduction of theexogenous copy of a nucleic acid encoding a PD-L1.

In one embodiment of any one of the population of HSCs or compositioncomprising a population of HSCs, the cells are cryopreserved prior touse, for example, use in the treatment of an autoimmune disease or fordeliberate/intentional suppression of an immune response or the immunesystem in a subject.

In one embodiment of any one of the population of HSCs or compositioncomprising a population of HSCs, the population of modified HSCs areproduced by a method comprising contacting a sample of HSCs with avector carrying an exogenous copy of a nucleic acid encoding a PD-L1 tomodify the HSCs to produce a population of modified HSCs cells thatexpress PD-L1.

In one embodiment of any one of the population of HSCs or compositioncomprising a population of HSCs, the method further comprises ex vivoculturing to expand the resultant modified cells from the contactingwith the vector.

In one embodiment of any one of the population of HSCs or compositioncomprising a population of HSCs, the method further comprisesestablishing the expression of PD-L1 on the modified HSCs.

In one embodiment of any one of the population of HSCs or compositioncomprising a population of HSCs, the method further comprisesestablishing that there is at least one fold increase in the number ofPD-L1⁺ expressing cells compared to non-modified cells.

In one embodiment of any one of the composition comprising a populationof HSCs described, the composition further comprises at least anadditional immunosuppression therapy agent or drug.

In one embodiment of any one of the composition comprising a populationof HSCs described, the composition further comprises a pharmaceuticallyacceptable carrier. The carrier is preferable not cell or tissue culturemedia.

In one embodiment of any one of the composition comprising a populationof HSCs described, the composition further comprises serum or plasma.

In one embodiment, provided herein is an ex vivo method of producing apopulation of modified, PD-L1⁺ expressing HSCs, the method comprisingcontacting a sample of HSCs with a vector carrying an exogenous copy ofa nucleic acid encoding a PD-L1 to modify the HSCs whereby the exogenouscopy of a nucleic acid is introduced into the HSCs thereby producing apopulation of modified HSCs cells expressing PD-L1.

In one embodiment of any one of the ex vivo method described, the methodfurther comprises ex vivo culturing of the resultant modified cells fromthe contacting with the vector carrying an exogenous copy of a nucleicacid encoding a PD-L1.

In one embodiment of any one of the ex vivo method described, the methodfurther comprises establishing the expression of PD-L1 on the modifiedHSCs.

In one embodiment of any one of the ex vivo method described, the methodfurther comprises comprises establishing that there is at least one foldincrease in the number of PD-L1⁺ expressing cells compared tonon-modified cells.

In one embodiment of any one of the ex vivo method described, the sampleof HSC is obtained from the bone marrow, umbilical cord, amniotic fluid,chorionic villi, cord blood, placental blood or peripheral blood.

In one embodiment of any one of the ex vivo method described, the sampleof HSC is obtained from mobilized peripheral blood, e.g., mobilized bygranulocyte colony stimulating factor (G-CSF).

In one embodiment of any one of the ex vivo method described, the sampleof HSCs is obtained from a healthy individual.

In one embodiment of any one of the ex vivo method described, the sampleof HSCs is obtained from an individual with a diagnosed disease ordisorder.

In one embodiment of any one of the ex vivo method described, thediagnosed disease or disorder is an autoimmune disease or disorder.

In one embodiment of any one of the ex vivo method described, theautoimmune disease or disorder is T1D.

In one embodiment of any one of the ex vivo method described, the sampleof HSCs is obtained from an individual who has newly been diagnosed withT1D.

In one embodiment of any one of the ex vivo method described, the sampleof HSCs is obtained from an individual who has newly been detected tohave self-autoantibodies associated with T1D, e.g., GAD65 autoantibody,and islet antigen 2 autoantibody.

In one embodiment of any one of the ex vivo method described, the vectoris viral vector.

In one embodiment of any one of the ex vivo method described, the viralvector is a lentiviral vector, an avian virus vector or anadeno-associated virus.

In one embodiment of any one of the ex vivo method described, thenucleic acid is a cDNA.

In one embodiment of any one of the ex vivo method described, thenucleic acid is a genomic DNA.

In one embodiment of any one of the ex vivo method described, thenucleic acid is integrated into the genome of the cells.

In one embodiment, provided herein is a method of treating an autoimmunedisorder or suppressing an immune response in a subject in need thereof,the method comprising administering to a subject a compositioncomprising the hematopoietic stem cells described herein.

In one embodiment, provided herein is a method of preventing or treatingan autoimmune disorder or suppressing an immune response in a subject inneed thereof, the method comprising providing a population of HSCs; exvivo contacting the sample of HSCs with prostaglandin E2 (PGE₂) at 10 μMconcentration for about 60 minutes at 37° C.; removing the PGE₂ after 60minutes, thereby producing a population of PD-L1⁺ expressing HSCs;transplanting the population of PD-L1⁺ expressing HSCs into a recipientsubject, thereby modulating the immune response in the recipientsubject.

In one embodiment, provided herein is a method of delaying the onset ofT1D in a subject in need thereof, the method comprising providing apopulation of HSCs; ex vivo contacting the sample of HSCs withprostaglandin E2 (PGE₂) at 10 μM concentration for about 60 minutes at37° C.; removing the PGE₂ after 60 minutes, thereby producing apopulation of PD-L1⁺ expressing HSCs; transplanting the population ofPD-L1⁺ expressing HSCs into a recipient subject, thereby modulating theimmune response in the recipient subject. In one embodiment, the subjectis at risk of developing T1D. In one embodiment, the subject isasymptomatic for T1D and is not hyperglycemia. For example, thesubject's a blood sugar level is not higher than 11.1 mmol/l (200mg/dl). In one embodiment, the subject is has recently been detected tohave self-autoantibodies associated with T1D, e.g., ICA, IAA and 1A-2A.

In one embodiment, provided herein is a method of preventing or delayingan allogenic tissue/organ rejection in a subject in need thereof, themethod comprising providing a population of HSCs; ex vivo contacting thesample of HSCs with prostaglandin E2 (PGE₂) at 10 μM concentration forabout 60 minutes at 37° C.; removing the PGE₂ after 60 minutes, therebyproducing a population of PD-L1⁺ expressing HSCs; transplanting thepopulation of PD-L1⁺ expressing HSCs into a recipient subject, therebymodulating the immune response in the recipient subject. In oneembodiment, the subject is an organ or tissue transplant recipient.

In one embodiment, provided herein is a method of preventing or treatingan autoimmune disorder or suppressing an immune response in a subject inneed thereof, the method comprising providing a population of HSCs; exvivo contacting the sample of HSCs with prostaglandin E2 (PGE₂) at 0.1μM concentration for at least 24 hours at 37° C.; removing the PGE₂,thereby producing a population of PD-L1⁺ expressing HSCs; transplantingthe population of PD-L1⁺ expressing HSCs into a recipient subject,thereby modulating the immune response in the recipient subject.

In one embodiment, provided herein is a method of delaying the onset ofT1D in a subject in need thereof, the method comprising providing apopulation of HSCs; ex vivo contacting the sample of HSCs withprostaglandin E2 (PGE₂) at 0.1 μM concentration for at least 24 hours at37° C.; removing the PGE₂, thereby producing a population of PD-L1⁺expressing HSCs; transplanting the population of PD-L1⁺ expressing HSCsinto a recipient subject, thereby modulating the immune response in therecipient subject. In one embodiment, the subject is at risk ofdeveloping T1D. In one embodiment, the subject is asymptomatic for T1Dand is not hyperglycemia. For example, the subject's a blood sugar levelis not higher than 11.1 mmol/l (200 mg/dl). In one embodiment, thesubject is has recently been detected to have self-autoantibodiesassociated with T1D, e.g., ICA, IAA and 1A-2A.

In one embodiment, provided herein is a method of preventing or delayingan allogenic tissue/organ rejection in a subject in need thereof, themethod comprising providing a population of HSCs; ex vivo contacting thesample of HSCs with prostaglandin E2 (PGE₂) at 0.1 μM concentration forat least 24 hours at 37° C.; removing the PGE₂, thereby producing apopulation of PD-L1⁺ expressing HSCs; transplanting the population ofPD-L1⁺ expressing HSCs into a recipient subject, thereby modulating theimmune response in the recipient subject. In one embodiment, the subjectis an organ or tissue transplant recipient.

In one embodiment, provided herein is a method of preventing or treatingan autoimmune disorder or suppressing an immune response in a subject inneed thereof, the method comprising: providing a population of HSCs; exvivo contacting the sample of HSCs with a vector carrying an exogenouscopy of a nucleic acid encoding a PD-L1; ex vivo culturing the resultantmodified cells from the contacting; establishing the expression of PD-L1on the modified HSCs, thereby producing a population of modified HSCscells expressing PD-L1, transplanting said population of PD-L1⁺expressing HSCs into a recipient subject, thereby modulating the immuneresponse in the recipient subject.

In one embodiment, provided herein is a method of delaying the onset ofT1D in a subject in need thereof, the method comprising: providing apopulation of HSCs; ex vivo contacting the sample of HSCs with a vectorcarrying an exogenous copy of a nucleic acid encoding a PD-L1; ex vivoculturing the resultant modified cells from the contacting; establishingthe expression of PD-L1 on the modified HSCs, thereby producing apopulation of modified HSCs cells expressing PD-L1, transplanting saidpopulation of PD-L1⁺ expressing HSCs into a recipient subject, therebymodulating the immune response in the recipient subject.

In one embodiment, provided herein is a method of preventing or delayingan allogenic tissue/organ rejection in a subject in need thereof, themethod comprising: providing a population of HSCs; ex vivo contactingthe sample of HSCs with a vector carrying an exogenous copy of a nucleicacid encoding a PD-L1; ex vivo culturing the resultant modified cellsfrom the contacting; establishing the expression of PD-L1 on themodified HSCs, thereby producing a population of modified HSCs cellsexpressing PD-L1, transplanting said population of PD-L1⁺ expressingHSCs into a recipient subject, thereby modulating the immune response inthe recipient subject.

In one embodiment of any one of the method described, the autoimmunedisorder is T1D.

In one embodiment of any one of the method described, the population ofHSCs provided is autologous to the recipient subject. In one embodiment,the subject is newly diagnosed with T1D. In another embodiment, thesubject is newly been detected to have self-autoantibodies associatedwith T1D, e.g., GAD65 autoantibody, and islet antigen 2 autoantibody.

In one embodiment of any one of the method described, the population ofHSCs provided is non-autologous and allogenic to the recipient subject.

In one embodiment of any one of the method described, the population ofHSCs provided is non-autologous and xenogeneic to the recipient subject.

In one embodiment of any one of the method described, the population ofHSCs provided is obtained from the bone marrow, umbilical cord, amnioticfluid, chorionic villi, cord blood, placental blood or peripheral blood.

In one embodiment of any one of the method described, the population ofHSCs provided is obtained from mobilized peripheral blood.

In one embodiment of any one of the ex vivo method described, thepopulation of HSCs provided is obtained from a healthy individual.

In one embodiment of any one of the ex vivo method described, thepopulation of HSCs provided is obtained from an individual with adiagnosed disease or disorder.

In one embodiment of any one of the ex vivo method described, thediagnosed disease or disorder is an autoimmune disease or disorder.

In one embodiment of any one of the ex vivo method described, theautoimmune disease or disorder is T1D.

In one embodiment of any one of the ex vivo method described, thepopulation of HSCs provided is obtained from an individual who has newlybeen diagnosed with T1D.

In one embodiment of any one of the ex vivo method described, thepopulation of HSCs provided is obtained from an individual who has newlybeen detected to have self-autoantibodies associated with T1D, e.g.,GAD65 autoantibody, and islet antigen 2 autoantibody.

In one embodiment of any one of the method described, the population ofHSCs provided is at the minimum CD 34⁺.

In one embodiment of any one of the method described, the population ofHSCs provided is at the minimum CD 34⁺ and Lin⁻.

In another embodiment of any one of the method described, the populationof HSCs provided is CD34⁺, CD59⁺, Thy1/CD90⁺, CD38^(lo/−), andC-kit/CD117⁺.

In one embodiment of any one of the method described, the population ofHSCs provided is CD34⁺-selected HSCs. In another embodiment, the HSCsare negatively selected against CD38. That is, only CD38^(lo/−) cellsare selected. In another embodiment, the HSCs are selected for CD34⁺ andCD38^(lo/−).

In one embodiment of any one of the method described, the PGE₂stimulated HSCs are also treated with steroids such as dexamethasome exvivo, prior to use in implantation into the recipient.

In one embodiment of any one of the method described, prior to thetransplantation into the recipient subject, the population of HSCs arecryopreserved after the removal of excess PGE₂ or cryopreserved after exvivo culturing to expand the population of HSCs post-transfection withthe vector carrying an exogenous copy of a nucleic acid encoding aPD-L1.

In one embodiment of any one of the method described, prior to thetransplantation into the recipient subject, the population of HSCs areculture expanded ex vivo after the removal of excess PGE₂ or aftertransfection with a vector the vector carrying an exogenous copy of anucleic acid encoding a PD-L1.

In one embodiment of any one of the method described, the method furthercomprising identifying a recipient subject having an autoimmune diseaseor disorder or an individual who is an organ or bone marrow transplantrecipient.

In one embodiment of any one of the method described, the method furthercomprising selecting a recipient subject having an autoimmune disease ordisorder or an individual who is an organ or bone marrow transplantrecipient.

In one embodiment of any one of the method described, the method furthercomprising identifying a recipient subject in need of the suppression ofan immune response or immune system or an individual who is an organ orbone marrow transplant recipient.

In one embodiment of any one of the method described, the method furthercomprising selecting a recipient subject in need of the suppression ofan immune response or immune system. For example, an individual who isan organ or bone marrow transplant recipient.

In one embodiment of any one of the method described, the method furthercomprising identifying a subject at risk of developing T1D. For example,a subject who is newly been detected to have self-autoantibodiesassociated with T1D, e.g., GAD65 autoantibody, and islet antigen 2autoantibody.

In one embodiment of the population of HSCs, the ex vivo method, thecomposition or the treatment method described herein, the PGE₂ thatstimulates PD-L1 expression in the HSCs is 16,16-Dimethyl prostaglandinE2 (dmPGE₂).

Definitions

As used herein, the term “nucleic acid” when used in reference toencoding a PD-L1 refers to refers to deoxyribonucleotides (DNA) orribonucleotides (RNA) and polymers thereof (“polynucleotides”) in eithersingle- or double-stranded form. Unless specifically limited, the termencompasses nucleic acids containing known analogues of naturalnucleotides that have similar binding properties as the referencenucleic acid and are metabolized in a manner similar to naturallyoccurring nucleotides. Unless otherwise indicated, a particular nucleicacid molecule/polynucleotide also implicitly encompasses conservativelymodified variants thereof (e.g. degenerate codon substitutions) andcomplementary sequences as well as the sequence explicitly indicated.Specifically, degenerate codon substitutions may be achieved bygenerating sequences in which the third position of one or more selected(or all) codons is substituted with mixed-base and/or deoxyinosineresidues (Batzer et al., Nucleic Acid Res. 19: 5081 (1991); Ohtsuka etal., J. Biol. Chem. 260: 2605-2608 (1985); Rossolini et al., Mol. Cell.Probes 8: 91-98 (1994)). Nucleotides are indicated by their bases by thefollowing standard abbreviations: adenine (A), cytosine (C), thymine(T), and guanine (G).

In some embodiments, as used herein, the term “genetically engineered,”“genetically modified” or “modified” refers to the addition, deletion,or modification of the genetic material in a cell. In some embodiments,the terms, “genetically modified cells” and “modified cells,” are usedinterchangeably. In other embodiments, “modified cells” refer topharmacologically PGE₂-stimulated HSCs or pharmacologicallyPGE₂-modified HSCs that express PD-L1 compared to prior to thestimulation.

In one embodiment, the term “non-modified HSCs” refers to HSCs that donot carry exogenous copies of a nucleic acid encoding a PD-L1. Inanother embodiment, the term “non-modified HSCs” refers to HSCs thathave not been ex vivo pharmacologically stimulated by PGE₂.

As used herein, the term “exogenous copy” in the context of a codingnucleic acid refers to an extra copy of the coding nucleic acid that isnot the original copy of the gene found in the genome of the HSCs. Theextra copy of the coding nucleic acid is typically introduced into thecells. For example, the extra copy is carried in a vector. The extracopy may be integrated into the genome of the cells.

As used herein, the term “coding” or “encoding” in the context of anucleic acid encoding a PD-L1 means the nucleic acid containsinstruction or information therein to specify the genetic code for aprotein, e.g., the cell surface protein PD-L1. The instruction orinformation in a coding nucleic acid can be transcribe and translated tothe encoded protein.

As used herein, the term “cDNA” refers to complementary DNA that isdouble-stranded DNA synthesized from a messenger RNA (mRNA) template ina reaction catalysed by the enzyme reverse transcriptase. The cDNA lacksintrons.

As used herein, a genomic DNA encoding a PD-L1 means the copy of thegene as found in the genome of a cell. The genomic DNA encoding a PD-L1would include introns and other regulatory sequences in addition to thecoding exons.

As used herein, the term “integrated” when used in the context of thenucleic acid encoding a PD-L1 means that the nucleic acid is insertedinto the genome or the genomic sequences of a cell. When integrated, theintegrated nucleic acid is replicated and divided into the daughterdividing cells in the same manner as the original genome of the cell.

As used herein, the term “vector”, when used in the context of carryingan exogenous copy of a nucleic acid encoding a PD-L1 vector, refersbroadly to a nucleic acid construct designed for delivery an exogenousnucleic acid to a host cell or transfer between different host cells. Inone embodiment, a vector can be viral or non-viral. In otherembodiments, a vector refers to any plasmid, phagemid or virus encodingan exogenous nucleic acid. In other embodiments, the term is also beconstrued to include non-plasmid, non-phagemid and non-viral compoundswhich facilitate the transfer of nucleic acid into virions or cells,such as, for example, poly-lysine compounds and the like. The vector maybe a viral vector that is suitable as a delivery vehicle for delivery ofthe nucleic acid, or mutant thereof, to a cell, or the vector may be anon-viral vector which is suitable for the same purpose. Examples ofviral and non-viral vectors for delivery of DNA to cells and tissues arewell known in the art and are described, for example, in Ma et al.(1997, Proc. Natl. Acad. Sci. U.S.A. 94: 12744-12746). Examples of viralvectors include, but are not limited to, a recombinant Vaccinia virus, arecombinant adenovirus, a recombinant retrovirus, a recombinantadeno-associated virus, a recombinant avian pox virus, and the like(Cranage et al., 1986, EMBO J. 5: 3057-3063; International PatentApplication No. WO94/17810, published Aug. 18, 1994; InternationalPatent Application No. WO94/23744, published Oct. 27, 1994). Examples ofnon-viral vectors include, but are not limited to, liposomes, polyaminederivatives of DNA, and the like.

As used herein, the term “viral vector” is used according to itsart-recognized meaning. It refers to a nucleic acid vector constructthat includes at least one element of viral origin and may be packagedinto a viral vector particle. The vector may be utilized for the purposeof transferring DNA, RNA or other nucleic acids into cells either invitro or in vivo. Numerous forms of viral vectors are known in the art.

As used herein, the term “lentivirus” refers to a group (or genus) ofretroviruses that give rise to slowly developing disease. Virusesincluded within this group include HIV (human immunodeficiency virus;including HIV type 1, and HIV type 2), the etiologic agent of the humanacquired immunodeficiency syndrome (AIDS); visna-maedi, which causesencephalitis (visna) or pneumonia (maedi) in sheep, the caprinearthritis-encephalitis virus, which causes immune deficiency, arthritis,and encephalopathy in goats; equine infectious anemia virus, whichcauses autoimmune hemolytic anemia, and encephalopathy in horses; felineimmunodeficiency virus (FIV), which causes immune deficiency in cats;bovine immune deficiency virus (BIV), which causes lymphadenopathy,lymphocytosis, and possibly central nervous system infection in cattle;and simian immunodeficiency virus (SIV), which cause immune deficiencyand encephalopathy in sub-human primates. Diseases caused by theseviruses are characterized by a long incubation period and protractedcourse. Usually, the viruses latently infect monocytes and macrophages,from which they spread to other cells. HIV, FIV, and SIV also readilyinfect T lymphocytes, i.e., T-cells.

As used herein, the term “lentiviral vector” refers to a vector having anucleic acid vector construct that includes at least one element oflentivirus origin. Lentiviral vectors of the disclosure include, but arenot limited to, human immunodeficiency virus (e.g., HIV-1, HIV-2),feline immunodeficiency virus (FIV), simian immunodeficiency virus(SIV), bovine immunodeficiency virus (BIV), and equine infectious anemiavirus (EIAV). These vectors can be constructed and engineered usingart-recognized techniques to increase their safety for use in therapyand to include suitable expression elements and therapeutic genes.

As used herein, the term “autoimmune disease” or “autoimmune disease ordisorder” herein is a disease or disorder arising from and directedagainst an individual's own tissues or a co-segregate or manifestationthereof or resulting condition therefrom.

Auto-immune related diseases and disorders arise from an overactiveand/or abnormal immune response of the body against substances(autoantigens) and tissues normally present in the body, otherwise knownas self or autologous substance. This dysregulated inflammatory reactioncauses an exaggerated response by macrophages, granulocytes, and/orT-lymphocytes leading to abnormal tissue damage and cell death.Subsequent loss of function is associated with inflammatory tissuedamage.

Autoantigens, as used herein, are endogenous proteins or fragmentsthereof that elicit this pathogenic immune response. Autoantigen can beany substance or a portion thereof normally found within a mammal that,in an autoimmune disease, becomes the primary (or a primary) target ofattack by the immune system. The term also includes antigenic substancesthat induce conditions having the characteristics of an autoimmunedisease when administered to mammals. Additionally, the term includespeptic subclasses consisting essentially of immunodominant epitopes orimmunodominant epitope regions of autoantigens. Immunodominant epitopesor regions in induced autoimmune conditions are fragments of anautoantigen that can be used instead of the entire autoantigen to inducethe disease. In humans afflicted with an autoimmune disease,immunodominant epitopes or regions are fragments of antigens specific tothe tissue or organ under autoimmune attack and recognized by asubstantial percentage (e.g. a majority though not necessarily anabsolute majority) of autoimmune attack T-cells.

Autoantigens that are known to be associated with autoimmune diseaseinclude myelin proteins with demyelinating diseases, e.g. multiplesclerosis and experimental autoimmune myelitis; collagens and rheumatoidarthritis; insulin, proinsulin, glutamic acid decarboxylase 65 (GAD65);islet cell antigen (ICA512; ICA12) with insulin dependent diabetes.

A common feature in a number of autoimmune related diseases andinflammatory conditions is the involvement of pro-inflammatory CD4+ Tcells. These T cells are responsible for the release of inflammatory,Th1 type cytokines. Cytokines characterized as Th1 type includeinterleukin 2 (IL-2), y-interferon, TNFα and IL-12. Suchpro-inflammatory cytokines act to stimulate the immune response, in manycases resulting in the destruction of autologous tissue. Cytokinesassociated with suppression of T cell response are the Th2 type, andinclude IL-10, IL-4 and TGF-β. It has been found that Th1 and Th2 type Tcells may use the identical antigen receptor in response to animmunogen; in the former producing a stimulatory response and in thelatter a suppressive response.

In one embodiment, as used herein, the term “hematopoietic stem cell” or“HSC” refers to a stem cell that give rise to all the blood cell typesof the three hematopoietic lineages, erythroid, lymphoid, and myeloid.These cell types include the myeloid (monocytes and macrophages,neutrophils, basophils, eosinophils, erythrocytes,megakaryocytes/platelets, dendritic cells), and the lymphoid lineages(T-cells, B-cells, NK-cells). In one embodiment, the term “hematopoieticstem cell” or “HSC” refers to a stem cell that have the following cellsurface markers: CD34⁺, CD59⁺, Thy1/CD90⁺, CD38^(lo/−), andC-kit/CD117⁺. In one embodiment, the term “hematopoietic stem cell” or“HSC” refers to a stem cell that is at least CD34⁺. In one embodiment,the term “hematopoietic stem cell” or “HSC” refers to a stem cell thatis at least CD38lo/⁻. In one embodiment, the term “hematopoietic stemcell” or “HSC” refers to a stem cell that is at least CD34⁺ andCD38^(lo/−). In one embodiment, the term “hematopoietic stem cell” or“HSC” refers to a stem cell that is at least lin⁻. In one embodiment,the term “hematopoietic stem cell” or “HSC” refers to a stem cell thatis at least CD34⁺ and lin⁻. In one embodiment, the term “hematopoieticstem cell” or “HSC” refers to a stem cell that is at least CD34⁺,CD38^(lo/−) and lin⁻. In one embodiment, the term “hematopoietic stemcell” or “HSC” refers to a stem cell that is at least CD34⁺ andC-kit/CD117⁺. In one embodiment, the term “hematopoietic stem cell” or“HSC” refers to a stem cell that is at least CD34⁺, CD38^(lo/−) andC-kit/CD117⁺. In another embodiment, as used herein, the term“hematopoietic stem cell” or “HSC” includes hematopoietic stem andprogenitor cells (HSPC).

In one embodiment, as used herein, the term “a progenitor cell” refersto refer to an immature or undifferentiated cell that has the potentiallater on to mature (differentiate) into a specific cell type, forexample, a blood cell, a skin cell, a bone cell, or a hair cells. Aprogenitor cell also can proliferate to make more progenitor cells thatare similarly immature or undifferentiated.

Cells of the disclosure can be autologous/autogeneic (“self”) ornon-autologous (“non-self,” e.g., allogeneic, syngeneic or xenogeneic).“Autologous,” as used herein, refers to cells from the same subject.

“Allogeneic,” as used herein, refers to cells of the same species thatdiffer genetically to the cell in comparison.

“Syngeneic,” as used herein, refers to cells of a different subject thatare genetically identical to the cell in comparison.

“Xenogeneic,” as used herein, refers to cells of a different species tothe cell in comparison. In preferred embodiments, the cells of thedisclosure are allogeneic.

An “isolated cell” refers to a cell that has been obtained from an invivo tissue or organ and is substantially free of extracellular matrix.

A “subject,” as used herein, includes any animal that possess ahematopoietic system, an immune system and HSCs. In one embodiment, asubject includes any animal that exhibits symptoms of a disease,disorder, or condition of the immune system, e.g., autoimmune disease,that can be treated with the HSCs described herein, and methodscontemplated herein. Suitable subjects (e.g., patients) includelaboratory animals (such as mouse, rat, rabbit, or guinea pig), farmanimals, and domestic animals or pets (such as a cat or dog). Non-humanprimates and, preferably, human patients, are included. Typical subjectsinclude animals that exhibit aberrant amounts (lower or higher amountsthan a “normal” or “healthy” subject) of one or more physiologicalactivities that can be modulated by the HSCs described herein, andmethods disclosed elsewhere herein. In another embodiment, the subjectis a human.

In one embodiment, as used herein “treatment” or “treating,” includesany beneficial or desirable effect on the symptoms or pathology of adisease or pathological condition, and may include even minimalreductions in one or more measurable markers of the disease or conditionbeing treated. In another embodiment, treatment can involve optionallyeither the reduction or amelioration of symptoms of the disease orcondition, or the delaying of the progression of the disease orcondition. “Treatment” does not necessarily indicate completeeradication or cure of the disease or condition, or associated symptomsthereof.

As used herein, “self-autoantibodies associated with T1D” refer to theautoantibodies that are markers of beta cell autoimmunity in type 1diabetes: Islet Cell Antibodies (ICA, against cytoplasmic proteins inthe beta cell), antibodies to Glutamic Acid Decarboxylase (GAD-65),Insulin Autoantibodies (IAA), and IA-2A, to protein tyrosinephosphatase.

As used herein, in one embodiment, the term “pharmaceuticallyacceptable” means approved by a regulatory agency of the Federal or astate government or listed in the U.S. Pharmacopeia or other generallyrecognized pharmacopeia for use in animals, and more particularly inhumans. Specifically, it refers to those compounds, materials,compositions, and/or dosage forms which are, within the scope of soundmedical judgment, suitable for use in contact with the tissues of humanbeings and animals without excessive toxicity, irritation, allergicresponse, or other problem or complication, commensurate with areasonable benefit/risk ratio.

The term “carrier” refers to a diluent, adjuvant, excipient, or vehiclewith which the therapeutic is administered. Such pharmaceutical carrierscan be sterile liquids, such as water and oils, including those ofpetroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil and the like. Water is a preferredcarrier when the pharmaceutical composition is administeredintravenously. Saline solutions and aqueous dextrose and glycerolsolutions can also be employed as liquid carriers, particularly forinjectable solutions. Suitable pharmaceutical excipients include starch,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, sodium stearate, glycerol monostearate, talc, sodium chloride,dried skim milk, glycerol, propylene, glycol, water, ethanol and thelike. The composition, if desired, can also contain minor amounts ofwetting or emulsifying agents, or pH buffering agents. Thesecompositions can take the form of solutions, suspensions, emulsion,tablets, pills, capsules, powders, sustained-release formulations, andthe like. The composition can be formulated as a suppository, withtraditional binders and carriers such as triglycerides. Oral formulationcan include standard carriers such as pharmaceutical grades of mannitol,lactose, starch, magnesium stearate, sodium saccharine, cellulose,magnesium carbonate, etc. Examples of suitable pharmaceutical carriersare described in Remington's Pharmaceutical Sciences, 18th Ed., Gennaro,ed. (Mack Publishing Co., 1990). The formulation should suit the mode ofadministration.

In one embodiment, “pharmaceutically acceptable carriers” exclude tissueculture medium. In another embodiment, “pharmaceutically acceptablecarriers” include serum or plasma. The serum or plasma can be derivedfrom human or the subject recipient.

The term “effective amount” means an amount of biologically activevector particles or PGE₂ concentration sufficient to provide successfultransduction of cells with the exogenous nucleic acid or to providesuccessful stimulation of PD-L1 expression in the cell respectively.

As used herein, the terms “administering,” refers to the placement ofthe HSCs described herein or the composition comprising the HSCsdescribed herein into a recipient subject by a method or route whichresults in at least partial localization of the HSCs at a desired site,or results in the proliferation, engraftment and/or differentiation ofthe HSCs to PD-L1 expressing progeny cells. The HSCs or the compositioncomprising the HSCs can be administered by any appropriate route whichresults in an effective treatment in the subject.

As used herein, the term “comprising” or “comprises” is used inreference to methods, and respective component(s) thereof, that areessential to the disclosure, yet open to the inclusion of unspecifiedelements, whether essential or not. The use of “comprising” indicatesinclusion rather than limitation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows that PD-L1 genetic deletion abrogates HSC immunomodulatoryproperties in vitro.

FIGS. 2A and 2B show that the percentage of peripheral PD-L1⁺ HSCs isreduced in NOD mice compared to B6.

FIG. 2C shows the confirmation of PD-L1 expression defect in NOD mice byPCR.

FIGS. 2D and 2E show that the murine PD-L1 defect on HSCs can beoverturned in vitro by pharmacologic approach. After 8 days of in vitroculture, an increase in the percentage of PD-L1⁺ KLS cells was evident.

FIGS. 3A and 3B show that the PD-L1⁺ HSCs are fewer in number in T1Dindividuals as compared to healthy individuals.

FIG. 3C shows the confirmation of PD-L1 defect, by PCR, in HSCs ofindividuals affected with T1D.

FIGS. 3D and 3E show that the human PD-L1 defect on HSCs can beoverturned in vitro by pharmacologic approach. After 7 days of in vitroculture, an increase in the percentage of PD-L1⁺ HSCs was evident.

FIGS. 4A and 4B show that PD-L1/PD-1 cross-linking with PIM2 delaysdiabetes onset in NOD mice (FIG. 4A) and prolongs islet survival postislet transplantation (inBALB/c into B6) (FIG. 4B).

FIGS. 5A and 5B show that the HSCs transduced with PDL1 cDNA bearinglentivirus become highly PDL1⁺ and once adoptively transferred intonewly diabetic NOD mice normalized glycemia. NOD untreated mice remainedhyperglycemic at >250 mg/dl.

FIG. 6 show the effect of PGE₂ on PDL1 expression on HSC.

FIG. 7 show that the murine PD-L1 transduced KLS cells revertedhyperglycemia in NOD mice.

FIG. 8A is a table summarizing the microarray analyses ofSca-1⁺Lineage⁻c-kit⁺HSCs from bone marrow of NOD and B6 mice showingthat genes were differentially expressed.

FIG. 8B is a Western Blot showing the reduced expression of PD-L1 inSca-1⁺Lineage⁻c-kit⁺HSCs from bone marrow of NOD compared to normal B6control mice.

FIG. 8C is a histogram summarizing the relative expression of PD-L1 inSca-1⁺Lineage⁻c-kit⁺HSCs from bone marrow of NOD compared to normal B6control mice, data obtained by Western blot analysis and quantitativemeasurements. Open histogram is NOD mice, closed histogram is C57BL/6mice.

FIG. 8D is a histogram summarizing the relative mRNA expression of PD-L1in Sca-1⁺Lineage⁻c-kit⁺HSCs from bone marrow of NOD compared to normalB6 control mice. Open histogram is NOD mice, closed histogram is C57BL/6mice.

FIGS. 8E and 8F show the FACS dot plots and the histograms of PD-L1⁺KLS: Sca-1⁺Lineage⁻c-kit⁺ cells from bone marrow of NOD compared tonormal B6 control mice. Open histogram is NOD mice, closed histogram isC57BL/6 mice.

FIGS. 8G and 8H show the FACS dot plots and the histograms of PD-L1⁺CD41⁻CD48⁻CD150⁺ cells from bone marrow of NOD compared to normal B6control mice. Open histogram is NOD mice, closed histogram is C57BL/6mice.

FIGS. 81 and 8K show the FACS dot plots and the histograms of PD-L1⁺ KL:Lineage⁻c-kit⁺ cells from bone marrow of NOD compared to normal B6control mice. Open histogram is NOD mice, closed histogram is C57BL/6mice.

FIGS. 8J and 8L show the FACS dot plots and the histograms of PD-L1⁺CD244⁻CD48⁻CD150⁺ cells from bone marrow of NOD compared to normal B6control mice. Open histogram is NOD mice, closed histogram is C57BL/6mice.

FIG. 9A shows the flow cytometric analysis of PD-L1 expression on KLcells extracted from NOD mice prior to transduction with PD-L1lentivirus, also known as wild type (WT) KL cells.

FIG. 9B shows the flow cytometric analysis of PD-L1 expression on KLcells from NOD mice after transduction with PD-L1 lentivirus, labeled asTg cells.

FIG. 9C shows the histogram summarizing the increased in PD-L1expression on KL cells from NOD mice after transduction with PD-L1lentivirus.

FIG. 9D shows the histogram summarizing the flow cytometric analysis ofINFγ production by CD4+ T cells extracted from NOD-BDC2.5 TCRtg micestimulated by BDC2.5 peptides in the presence of DCs and upon coculturewith KL cells and with PD-L1. Tg KL cells.

FIG. 9E shows the flow cytometric analysis of INFγ production by CD4+ Tcells extracted from NOD-BDC2.5 TCRtg mice stimulated by BDC2.5 peptidesin the presence of DCs and upon coculture with KL cells and with PD-L1.Tg KL cells in the presence of PD-L1 blocking/neutralizing Ab.

FIG. 9F shows the histogram summarizing the flow cytometric analysis ofINFγ production by CD4+ T cells extracted from NOD mice stimulated bysoluble anti-CD3/anti-CD28 upon coculture with KL cells and with PD-L1.Tg KL cells.

FIG. 9G shows the flow cytometric analysis of INFγ production by CD4+ Tcells extracted from NOD mice stimulated by soluble anti-CD3/anti-CD28upon coculture with KL cells and with PD-L1. Tg KL cells in the presenceof PD-L1 blocking/neutralizing Ab.

FIGS. 9H-9K are graphical representations of reversal of diabetes inNOD-Hyperglycemic treated with untransduced KL cells (FIG. 9K) andPD-L1.Tg KL cells (FIG. 9I) as demonstrated by blood glucose levelsfollowing administration of 3×10⁶ untransduced KL cells or PD-L1.Tg KLcells. No reversal was achieved with doxycycline (FIG. 9J); (FIG. 9H)Untreated group used as control.

FIGS. 10A-10F demonstrated that the PD-L1 defect in human HSCs from T1Dpatients as compared to healthy controls human subjects (HC).

FIGS. 10A-10B are representative flow cytometric analysis showing PD-L1expression in selected CD34⁺HSCs from healthy controls (HC) (FIG. 10A)and from type 1 diabetic individuals (T1D) (FIG. 10B).

FIG. 10C shows the bar graph related to the flow cytometric analysis inFIGS. 10A-10B, illustrating the defect in PD-L1 expression in T1D.

FIG. 10D is a representative Western-blot analysis showing reduced PD-L1expression in CD34+HSCs of T1D individual compared to HC.

FIG. 10E is a histogram summarizing the Western-blot analysis showingreduced PD-L1 expression in CD34⁺HSCs of T1D individual compared to HC.

FIG. 10F is a histogram summarizing the RT-PCR data for PD-L1 expressionin CD34⁺HSCs of T1D individual compared to HC.

FIG. 11 shows the effect of dual PGE₂ and dexamethasone-stimulated KLcells in normalizing hyperglycemia in NOD mice after the onset ofhyperglycemia. Each line represents the blood sugar of a test NOD mouse.The KL cells were stimulated ex vivo prior to implantation into therecipient mouse shortly after the onset of hyperglycemia.

FIG. 12 shows that mice treated with PGE₂-stimulated HSC have delayedislet allograft rejection. Similar strategy can be used in general toprevent and also treat allograft rejections.

DETAILED DESCRIPTION

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. It should beunderstood that this disclosure is not limited to the particularmethodology, protocols, and reagents, etc., described herein and as suchcan vary. The terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to limit the scope ofthe present disclosure, which is defined solely by the claims.

Definitions of common terms in molecular biology can be found in TheMerck Manual of Diagnosis and Therapy, 19th Edition, published by MerckSharp & Dohme Corp., 2011 (ISBN 978-0-911910-19-3), (2015 digital onlineedition at merckmanuals.com), Robert S. Porter et al. (eds.), TheEncyclopedia of Molecular Cell Biology and Molecular Medicine, publishedby Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); and Robert A.Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive DeskReference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8);Immunology by Werner Luttmann, published by Elsevier, 2006; Janeway'sImmunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), Taylor& Francis Limited, 2014 (ISBN 0815345305, 9780815345305); Lewin's GenesXI, published by Jones & Bartlett Publishers, 2014 (ISBN-1449659055);Michael Richard Green and Joseph Sambrook, Molecular Cloning: ALaboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., USA (2012) (ISBN 1936113414); Davis et al., BasicMethods in Molecular Biology, Elsevier Science Publishing, Inc., NewYork, USA (2012) (ISBN 044460149X); Laboratory Methods in Enzymology:DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); CurrentProtocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), JohnWiley and Sons, 2014 (ISBN 047150338X, 9780471503385), Current Protocolsin Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons,Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan,ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe,(eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737),the contents of which are all incorporated by reference herein in theirentireties. Further, unless otherwise required by context, singularterms shall include pluralities and plural terms shall include thesingular.

Unless otherwise stated, the present disclosure was performed usingstandard procedures known to one skilled in the art, for example, inMichael R. Green and Joseph Sambrook, Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,USA (2012); Davis et al., Basic Methods in Molecular Biology, ElsevierScience Publishing, Inc., New York, USA (1986); Current Protocols inMolecular Biology (CPMB) (Fred M. Ausubel, et al. ed., John Wiley andSons, Inc.), Current Protocols in Immunology (CPI) (John E. Coligan, et.al., ed. John Wiley and Sons, Inc.), Current Protocols in Cell Biology(CPCB) (Juan S. Bonifacino et. al. ed., John Wiley and Sons, Inc.),Culture of Animal Cells: A Manual of Basic Technique by R. Ian Freshney,Publisher: Wiley-Liss; 5th edition (2005), Animal Cell Culture Methods(Methods in Cell Biology, Vol. 57, Jennie P. Mather and David Barneseditors, Academic Press, 1st edition, 1998), Methods in Molecularbiology, Vol. 180, Transgenesis Techniques by Alan R. Clark editor,second edition, 2002, Humana Press, and Methods in Meolcular Biology,Vo. 203, 2003, Transgenic Mouse, edited by Marten H. Hofker and Jan vanDeursen, which are all herein incorporated by reference in theirentireties.

It should be understood that this disclosure is not limited to theparticular methodology, protocols, and reagents, etc., described hereinand as such may vary. The terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the present disclosure, which is defined solely by the claims.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used in connection with percentages willmean±1%.

All patents and publications identified are expressly incorporatedherein by reference for the purpose of describing and disclosing, forexample, the methodologies described in such publications that might beused in connection with the present disclosure. These publications areprovided solely for their disclosure prior to the filing date of thepresent application. Nothing in this regard should be construed as anadmission that the inventors are not entitled to antedate suchdisclosure by virtue of prior disclosures or for any other reason. Allstatements as to the date or representation as to the contents of thesedocuments is based on the information available to the applicants anddoes not constitute any admission as to the correctness of the dates orcontents of these documents.

The present disclosure relates to modified hematopoietic stem cells(HSCs), compositions comprising modified HSCs, methods of using thesemodified HSCs for treating autoimmune diseases and disorders and formodulating the immune system. The modified HSCs express the programmedcell death-1 receptor ligand (PD-L1) if the cells did not express PD-L1prior to the modification or the modified HSCs now express more PD-L1compared to prior to the modification. The modification is bytransducing an exogenous copy of a nucleic acid encoding PD-L1 tofacilitate PD-L1 protein expression in the transduced cell or bypharmacological re-programming of the HSCs with stimulation by PGE₂.

The disclosure described herein, in a preferred embodiment, does notconcern a process for cloning human beings, processes for modifying thegerm line genetic identity of human beings, uses of human embryos forindustrial or commercial purposes or processes for modifying the geneticidentity of animals which are likely to cause them suffering without anysubstantial medical benefit to man or animal, and also animals resultingfrom such processes.

In some embodiments, embodiments of the present disclosure are based onthe discovery that increasing PD-L1 expression in the HSCs of patientswith Type 1 diabetes (T1D) can alleviate the deficiencies in thepatients' immunoregulation. NOD mice and human T1D patients have reducednumber of PD-L1 expressing HSCs and the HSCs express lower amounts ofPD-L1. The decrease PD-L1 contribute to defects in the mice andpatients' ability to immunoregulation. Externally supplementing thisPD-L1 deficiency help recorrect this immunoregulation defect bypromoting immune tolerance.

Despite considerable effort to halt or delay the destruction ofbeta-cells in T1D, success remains elusive. Stem cells-based therapyusing mesenchymal stem cells and autologous hematopoietic stem celltransplantation (AHSCT) yield only short-term insulin-independence inNOD mice and T1D humans, and for only a select population of afflictedwith the disease. None of the stem cell-based therapies have not beenapplicable to pediatric patients. Moreover, certain stem cell basedtherapies present potential oncogenic concerns, especially for pediatricpatients.

Therefore, the problem to solve here is to provide a therapy that isapplicable to a larger population of T1D patients, both adults andpediatric patients, and a therapy that allows the patients to be longterm insulin-independent.

Previously, preclinical studies on the use of HSCs in NOD mice arelacking and primarily employ allogeneic HSCs. When allogeneic HSCs from0-gal transgenic donors were transplanted into NOD mice, diabetes onsetwas successfully preventing in all treated mice, but reversal wasobtained in only 1 out of 50 mice despite full hematopoieticengraftment. If a human is at high risk for developing T1D, perhapsadministering the PD-L1⁺ cells can delay the onset of the disease too.The inventors demonstrated that HSC immunological properties may belinked to the expression of the immunomodulatory molecule PD-L1 (alsoknown as CD274 or B7-H1). PD-L1 is the ligand of the inhibitory receptorprogrammed death 1 receptor (PD-1), which is expressed primarily onactivated T cells. Crosslinking between PD-L1 and PD-1 inhibits T cellsactivation and favor their exhaustion/apoptosis. PD-1 knockout micedevelop accelerated diabetes, and PD-1/PD-L1 signaling activates aninhibitory signal inducing T cell anergy.

The inventors found that there are fewer numbers of CD34⁺HSCs thatexpress the PD-L1 in patients with T1D compared to healthy humans. Thisdiscovery that was obtained by immunoflowcytometry was further confirmedby RT-PCR. There is about 3% PD-L1⁺/CD34⁺ HSCs for human patients withT1D compared to 14.5% PD-L1⁺/CD34⁺ HSCs for healthy humans. PD-L1 is animportant immunoregulator molecule in the immune system.

Furthermore, the inventors found that the HSCs from T1D patients weredefective in their immunoregulatory properties. When tested in ananti-CD3/CD28 ELISPOT immunoassay, the HSCs from these patients affectedby T1D were less capable of suppressing an immune response.

Therefore, increasing or stimulating PD-L1 expression in HSCs derivedfrom patients affected by T1D or other autoimmune disorders, and/orproviding PD-L1 expressing HSCs to these individuals represent usefultherapeutic strategies in treating autoimmune diseases and disorders,and for modulating the immune system. The inventors have discovered thatex vivo incubating the HSCs derived from T1D patients with prostaglandinE2 (PGE₂) stimulates expression of PD-L1 in the HSCs. In addition,transfecting an exogenous nucleic acid that codes for PD-L1 into HSCspromotes the expression of PD-L1 in the transfected/transduced HSCs.

PD-L1⁺ Expressing Hematopoietic Stem Cells (HSCs) and CompositionsThereof.

Accordingly, in one embodiment, provided herein is an ex vivo method ofproducing a population of modified, PD-L1⁺ expressing HSCs where themodified HSC cells carry an exogenous copy of a nucleic acid encoding aprogrammed cell death-1 receptor ligand (PD-L1), the method comprisingcontacting a sample of HSCs with a vector carrying an exogenous copy ofa nucleic acid encoding a PD-L1 to modify the HSCs, whereby theexogenous copy of a nucleic acid is introduced into the HSCs, therebyproducing a population of modified HSCs cells expressing PD-L1. In oneembodiment, the method further comprises establishing the expression ofPD-L1 on the resultant modified HSCs. In another embodiment, the methodfurther comprises ex vivo culturing the resultant modified cells aftercontact with the vector and/or ex vivo culturing the resultant modifiedcells after establishing the expression of PD-L1 on the resultantmodified HSCs. The culturing expands the number of modified cellsavailable for therapy. In one embodiment, the sample of HSCs can beculture expanded prior to contacting with the vector described.

In one embodiment, provided herein is an ex vivo method of producing apopulation of modified, PD-L1+ expressing HSCs where the modified HSCcells carry an exogenous copy of a nucleic acid encoding a PD-L1, themethod comprising (a) contacting a sample of HSCs with a vector carryingan exogenous copy of a nucleic acid encoding a PD-L1 to modify the HSCswhereby the exogenous copy of a nucleic acid is introduced into theHSCs; and (b) establishing the expression of PD-L1 on the resultantmodified HSCs, thereby producing a population of modified HSCs cellsexpressing PD-L1. In one embodiment, the method further comprises exvivo culturing the resultant modified cells after contact with thevector and/or ex vivo culturing the resultant modified cells afterestablishing the expression of PD-L1 on the resultant modified HSCs. Theculturing expands the number of modified cells available for therapy. Inone embodiment, the sample of HSCs can be culture expanded prior tocontacting with the vector described.

In one embodiment, provided herein is an ex vivo method of producing apopulation of modified, PD-L1+ expressing HSCs where the modified HSCcells carry an exogenous copy of a nucleic acid encoding a PD-L1, themethod comprising (a) contacting a sample of HSCs with a vector carryingan exogenous copy of a nucleic acid encoding a PD-L1 to modify the HSCswhereby the exogenous copy of a nucleic acid is introduced into theHSCs; (b) ex vivo culturing the resultant modified cells from thecontacting with the vector; and (c) establishing the expression of PD-L1on the resultant modified HSCs, thereby producing a population ofmodified HSCs cells expressing PD-L1. The culturing expands the numberof modified cells available for therapy. In one embodiment, the sampleof HSCs can be culture expanded prior to contacting with the vectordescribed.

In one embodiment, provided herein is a population of modified HSCswhere the modified HSCs carry an exogenous copy of a nucleic acidencoding a PD-L1. The modified HSCs express more PD-L1 compared tonon-modified cells not carrying an exogenous copy of a nucleic acidencoding a PD-L1.

In one embodiment, provided herein is a population of modified HSCs,wherein the cells are produced by a method comprising contacting asample of HSCs with a vector carrying an exogenous copy of a nucleicacid encoding a PD-L1 to modify the HSCs whereby the exogenous copy of anucleic acid is introduced into the HSCs, thereby producing a populationof modified HSCs cells expressing PD-L1. In one embodiment, the sampleof HSCs comprises non-modified HSCs. In one embodiment, non-modifiedHSCs do not carry exogenous copies of a nucleic acid encoding a PD-L1.In one embodiment, the method further comprises establishing theexpression of PD-L1 on the resultant modified HSCs. In anotherembodiment, the method further comprises ex vivo culturing the resultantmodified cells after contact with the vector and/or ex vivo culturingthe resultant modified cells after establishing the expression of PD-L1on the resultant modified HSCs. The culturing expands the number ofmodified cells available for therapy. In one embodiment, the sample ofHSCs can be culture expanded prior to contacting with the vectordescribed.

In one embodiment, provided herein is a population of modified HSCs,wherein the cells are produced by a method comprising (a) contacting asample of HSCs with a vector carrying an exogenous copy of a nucleicacid encoding a PD-L1 to modify the HSCs whereby the exogenous copy of anucleic acid is introduced into the HSCs; and (b) establishing theexpression of PD-L1 on the modified HSCs, thereby producing a populationof modified HSCs cells expressing PD-L1. In one embodiment, the methodfurther comprises ex vivo culturing the resultant modified cells aftercontact with the vector and/or ex vivo culturing the resultant modifiedcells after establishing the expression of PD-L1 on the resultantmodified HSCs. The culturing expands the number of modified cellsavailable for therapy. In one embodiment, the sample of HSCs can beculture expanded prior to contacting with the vector described.

In one embodiment, provided herein is a population of modified HSCs,wherein the cells are produced by a method comprising (a) contacting asample of HSCs with a vector carrying an exogenous copy of a nucleicacid encoding a PD-L1 to modify the HSCs whereby the exogenous copy of anucleic acid is introduced into the HSCs; (b) ex vivo culturing theresultant modified cells from the contacting; and (c) establishing theexpression of PD-L1 on the modified HSCs, thereby producing a populationof modified HSCs cells expressing PD-L1. The culturing expands orincreases the number of modified cells available for therapy. In oneembodiment, the sample of HSCs can be culture expanded prior tocontacting with the vector described.

In one embodiment, the modified HSCs are engineered modified cells,engineered to carrying an exogenous copy of a nucleic acid encoding aPD-L1 in the cell. These engineered HSCs express PD-L1 compared HSCs notcarrying an exogenous copy of a nucleic acid encoding a PD-L1. In oneembodiment, these engineered HSCs express more PD-L1 compared HSCs notcarrying an exogenous copy of a nucleic acid encoding a PD-L1.

In another embodiment, provided herein is an ex vivo method ofstimulating the expression of PD-L1 in a population of HSCs, the methodcomprising (a) contacting a sample of HSCs with prostaglandin E2 (PGE₂)at 10 μM concentration for about 60 min at 37° C.; (b) washing thecontacted cells to remove excess PGE₂, and (c) establishing theexpression of PD-L1 on the PGE₂-stimulated HSCs, thereby producing apopulation of PGE₂-stimulated HSCs cells expressing PD-L1.

In one embodiment, the PGE₂-stimulated HSC has increased PD-L1expression compared to non-PGE₂-stimulated HSC. In one embodiment, thePGE₂-stimulated HSC has at least 1% increased PD-L1 expression comparedto non-PGE₂-stimulated HSC. In other embodiments, the PGE₂-stimulatedHSC has at least 2%, at least 3%, at least 5%, at least 8%, at least10%, at least 15%, at least 20%, at least 25%, at least 30%, at least35%, at least 40%, at least 45%, at least 50%, at least 55%, at least60%, at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 95%, at least 1-fold, at least 2-fold, atleast 5-fold, at least 10 fold, at least 100 fold higher, at least1000-fold higher, or more increased PD-L1 expression compared tonon-PGE₂-stimulated HSC.

In another embodiment, provided herein is an ex vivo method ofstimulating the expression of PD-L1 in a population of HSCs, the methodcomprising (a) contacting a sample of HSCs with prostaglandin E2 (PGE₂)at 0.1 μM concentration for at least 24 hrs at 37° C.; (b) washing thecontacted cells to remove excess PGE₂, and (c) establishing theexpression of PD-L1 on the PGE₂-stimulated HSCs, thereby producing apopulation of PGE₂-stimulated HSCs cells expressing PD-L1.

In another embodiment, provided herein is an ex vivo method ofstimulating the expression of PD-L1 in a population of HSCs, the methodcomprising contacting a sample of HSCs with prostaglandin E2 (PGE₂) at0.1 μm concentration for at least 24 hrs at 37° C., thereby producing apopulation of PGE₂-stimulated HSCs cells expressing PD-L1.

In another embodiment, provided herein is an ex vivo method ofstimulating the expression of PD-L1 in a population of HSCs, the methodcomprising contacting a sample of HSCs with prostaglandin E2 (PGE₂) at10 μM concentration for about 60 min at 37° C., thereby producing apopulation of PGE₂-stimulated HSCs cells expressing PD-L1.

In one embodiment of the above described methods, the method furthercomprises washing the contacted cells to remove excess PGE₂. In oneembodiment, the method further comprises establishing the expression ofPD-L1 on the PGE₂-stimulated HSCs. In another embodiment, the methodfurther comprises ex vivo culturing of the PGE₂-stimulated HSCs aftercontact with PGE₂ and/or ex vivo culturing of the PGE₂-stimulated HSCsafter establishing the expression of PD-L1 on the PGE₂-stimulated HSCs.The culturing expands the number of modified cells available fortherapy. In one embodiment, the sample of HSCs can be culture expandedprior to contacting with PGE₂.

In another embodiment, provided herein is an ex vivo method ofstimulating the expression of PD-L1 in a population of HSCs, the methodcomprising (a) contacting a sample of HSCs with prostaglandin E2 (PGE₂)at 10 μM concentration for about 60 min at 37° C., and (b) establishingthe expression of PD-L1 on the PGE₂-stimulated HSCs, thereby producing apopulation of PGE₂-stimulated HSCs cells expressing PD-L1 therebyproducing a population of PGE₂-stimulated HSCs cells expressing PD-L1.The culturing expands the number of modified cells available fortherapy. In one embodiment, the sample of HSCs can be culture expandedprior to contacting with PGE₂.

In another embodiment, provided herein is an ex vivo method ofstimulating the expression of PD-L1 in a population of HSCs, the methodcomprising (a) contacting a sample of HSCs with prostaglandin E2 (PGE₂)at 0.1 μM concentration for at least 24 hrs at 37° C., and (b)establishing the expression of PD-L1 on the PGE₂-stimulated HSCs,thereby producing a population of PGE₂-stimulated HSCs cells expressingPD-L1 thereby producing a population of PGE₂-stimulated HSCs cellsexpressing PD-L1.

In another embodiment of the above described methods, the method furthercomprises ex vivo culturing of the PGE₂-stimulated HSCs after contactwith PGE₂ and/or ex vivo culturing of the PGE₂-stimulated HSCs afterestablishing the expression of PD-L1 on the PGE₂-stimulated HSCs. Theculturing expands the number of modified cells available for therapy. Inone embodiment, the sample of HSCs can be culture expanded prior tocontacting with PGE₂.

In one embodiment, provided herein is a population of PD-L1⁺ expressingHSCs wherein the cells are produced by a method comprising (a)contacting a sample of HSCs with PGE₂ at 10 μM concentration for about60 min at 37° C.; (b) washing the contacted cells to remove excess PGE₂,and (c) establishing the expression of PD-L1 on the contacted HSCs,thereby producing a population of HSCs cells expressing PD-L1.

In one embodiment, provided herein is a population of PD-L1⁺ expressingHSCs wherein the cells are produced by a method comprising (a)contacting a sample of HSCs with PGE₂ at 0.1 μM concentration for atleast 24 hrs at 37° C.; (b) washing the contacted cells to remove excessPGE₂, and (c) establishing the expression of PD-L1 on the contactedHSCs, thereby producing a population of HSCs cells expressing PD-L1.

In another embodiment of the above described methods, the method furthercomprises ex vivo culturing of the PGE₂-stimulated HSCs after contactwith PGE₂ and/or ex vivo culturing of the PGE₂-stimulated HSCs afterestablishing the expression of PD-L1 on the PGE₂-stimulated HSCs.

In one embodiment, provided herein is a population of PD-L1⁺ expressingHSCs where the cells have been stimulated to increase the expression ofendogenous PD-L1 by an ex vivo or in vivo or in vitro contact with PGE₂.

In one embodiment, provided herein is a population of modified HSCswhere the modified HSCs express more PD-L1 compared to non-modifiedcells that have not been stimulated or contacted with PGE₂.

In one embodiment, provided herein is a population of PD-L1⁺ expressingHSCs wherein the cells are produced by a method comprising contacting asample of HSCs with PGE₂ at 10 μM concentration for about 60 min at 37°C., thereby producing a population of HSCs cells expressing PD-L1.

In one embodiment, provided herein is a population of PD-L1⁺ expressingHSCs wherein the cells are produced by a method comprising contacting asample of HSCs with PGE₂ at 0.1 μM concentration for at least 24 hrs at37° C., thereby producing a population of HSCs cells expressing PD-L1.

In one embodiment of the above described methods or population of PD-L1⁺expressing HSCs, the method further comprises washing the contactedcells to remove excess PGE₂. In one embodiment, the method furthercomprises establishing the expression of PD-L1 on the PGE₂-stimulatedHSCs. In another embodiment, the method further comprises ex vivoculturing of the PGE₂-stimulated HSCs after contact with PGE₂ and/or exvivo culturing of the PGE₂-stimulated HSCs after establishing theexpression of PD-L1 on the PGE₂-stimulated HSCs.

In one embodiment, provided herein is a population of PD-L1⁺ expressingHSCs wherein the cells are produced by a method comprising (a)contacting a sample of HSCs with prostaglandin E2 (PGE₂) at 10 μMconcentration for about 60 min at 37° C., and (b) establishing theexpression of PD-L1 on the PGE₂-stimulated HSCs, thereby producing apopulation of PGE₂-stimulated HSCs cells expressing PD-L1 therebyproducing a population of PGE₂-stimulated HSCs cells expressing PD-L1.

In one embodiment, provided herein is a population of PD-L1⁺ expressingHSCs wherein the cells are produced by a method comprising (a)contacting a sample of HSCs with prostaglandin E2 (PGE₂) at 0.1 μMconcentration for at least 24 hrs 37° C., and (b) establishing theexpression of PD-L1 on the PGE₂-stimulated HSCs, thereby producing apopulation of PGE₂-stimulated HSCs cells expressing PD-L1 therebyproducing a population of PGE₂-stimulated HSCs cells expressing PD-L1.

In some embodiment of the above described methods or population ofPD-L1⁺ expressing HSCs, the HSCs are also contacted with a steroid suchas dexamethasone. In some embodiments, the HSCs are ex vivo contactedwith both PGE₂ and a steroid such as dexamethasone, ie., co-stimulatedsimultaneously with both PGE₂ and dexamethasone. For example,dexamethasone at 0.1 μM-100 μM, 0.1 μM, 0.5 WM, 1 WM, 5 μM, 10 WM, 20μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM or 100 μM.

In another embodiment of the above described methods or populations ofPD-L1⁺ expressing HSCs, the method further comprises ex vivo culturingof the PGE₂-stimulated HSCs after contact with PGE₂ and/or ex vivoculturing of the PGE₂-stimulated HSCs after establishing the expressionof PD-L1 on the PGE₂-stimulated HSCs.

In one embodiment of the described methods or populations of PD-L1⁺expressing HSCs herein, the sample of HSCs is cultured ex vivo in theabsence of PGE₂ before the addition/contact of PGE₂. The ex vivoculturing expands or increases the number of starting HSCs available forcontact and stimulation with PGE₂.

In one embodiment of the described methods or populations of PD-L1⁺expressing HSCs herein, the ex vivo culturing in the absence of PGE₂occurs for at least 48 hrs prior to the first/initial addition orcontact with PGE₂. The ex vivo culturing expands or increases the numberof starting HSCs available for contact and stimulation with PGE₂.

In one embodiment of the described methods or populations of PD-L1⁺expressing HSCs herein, the HSCs are in contact with PGE₂ in culture forat least 24 hrs. In other embodiments, the HSCs are in contact with PGE₂in culture for at least 36 hrs, at least 48 hrs, at least 60 hrs, atleast 72 hrs, at least 84 hrs, at least 96 hrs, at least 108 hrs, atleast 120 hrs, at least 132 hrs, at least 144 hrs, at least 156 hrs, atleast 168 hrs, at least 196 hrs and all intervening time in hoursbetween 24-196 hrs.

In another embodiment, the HSCs are in contact with PGE₂ in culture forup to eight days. In other embodiments, the HSCs are in contact withPGE₂ in culture for up to three days, for up to four days, for up tofive days, for up to six days and for up to seven days.

In other embodiments, the HSCs are in contact with PGE₂ in culture forabout 24 hrs, about 36 hrs, about 48 hrs, about 60 hrs, about 72 hrs,about 84 hrs, about 96 hrs, about 108 hrs, about 120 hrs, about 132 hrs,about 144 hrs, about 156 hrs, about 168 hrs, about 196 hrs and allintervening time in hours between 24-196 hrs.

In one embodiment, provided herein is a composition comprising apopulation of modified HSCs described herein, wherein the modified HSCsexpress PD-L1⁺. For example, the modified HSCs carry an exogenous copyof a nucleic acid encoding a PD-L1. In one embodiment, the compositionfurther comprises a pharmaceutically acceptable carrier. In oneembodiment, the pharmaceutically acceptable carrier does not includetissue culture media.

In one embodiment, provided herein is a pharmaceutical compositioncomprising a population of modified HSCs described herein and apharmaceutically acceptable carrier.

In one embodiment, provided herein is a composition comprising apopulation of PD-L1⁺ expressing HSCs described herein wherein the HSCsare modified HSCs carrying an exogenous copy of a nucleic acid encodinga PD-L1 or the HSCs are ex vivo stimulated to increase the expression ofendogenous PD-L1 by an ex vivo contact with PGE₂. In one embodiment, thecomposition further comprises a pharmaceutically acceptable carrier. Inone embodiment, the pharmaceutically acceptable carrier does not includetissue culture media.

In one embodiment, provided herein is a composition comprising apopulation of modified HSCs described herein for use in conjunction witha transplantation procedure, or for use with the treatment of anautoimmune disease or disorder, or for use in reducing or modulating animmune response, wherein the modified HSCs carry an exogenous copy of anucleic acid encoding a PD-L1 and express PD-L1. In one embodiment, thecomposition further comprises a pharmaceutically acceptable carrier. Inone embodiment, the pharmaceutically acceptable carrier does not includetissue culture media.

In one embodiment, provided herein is a composition comprising apopulation of PD-L1⁺ expressing HSCs described herein for use inconjunction with a transplantation procedure, or for use with thetreatment of an autoimmune disease or disorder, or for use in reducingor modulating an immune response, wherein the HSCs are modified HSCscarrying an exogenous copy of a nucleic acid encoding a PD-L1 or theHSCs are ex vivo stimulated to increase the expression of endogenousPD-L1 by an ex vivo contact with PGE₂. In one embodiment, thecomposition further comprises a pharmaceutically acceptable carrier. Inone embodiment, the pharmaceutically acceptable carrier does not includetissue culture media.

In one embodiment, provided herein is a composition comprising apopulation of modified HSCs described herein for manufacture of amedicament for use in conjunction with a transplantation procedure, orfor use with the treatment of an autoimmune disease or disorder, or foruse in reducing or modulating an immune response, wherein the modifiedHSCs carry an exogenous copy of a nucleic acid encoding a PD-L1 andexpress PD-L1. In one embodiment, the composition further comprises apharmaceutically acceptable carrier. In one embodiment, thepharmaceutically acceptable carrier does not include tissue culturemedia.

In one embodiment, provided herein is a composition comprising apopulation of PD-L1⁺ expressing HSCs described herein for manufacture ofa medicament for use in conjunction with a transplantation procedure, orfor use with the treatment of an autoimmune disease or disorder, or foruse in reducing or modulating an immune response, wherein the HSCs aremodified HSCs carrying an exogenous copy of a nucleic acid encoding aPD-L1 or the HSCs are ex vivo stimulated to increase the expression ofendogenous PD-L1 by an ex vivo contact with PGE₂. In one embodiment, thecomposition further comprises a pharmaceutically acceptable carrier. Inone embodiment, the pharmaceutically acceptable carrier does not includetissue culture media.

In one embodiment of the population of modified HSCs, the ex vivomethod, or the composition described herein, modified HSCs orPGE₂-contacted HSCs are further analyzed to establish the expression ofPD-L1 on the respective HSCs. Methods of determining PD-L1 expressionare known in the art, for example, by using immunoflowcytometry,fluorescence-activated cell sorting (FACS) or any immunoassays known inthe art, and by RT-PCR.

In one embodiment of the population of modified HSCs, the ex vivomethod, or the composition described herein, the modified HSCs areexpressing PD-L1. In one embodiment, there is at least one fold increasein the number of PD-L1⁺ expressing cells compared to control HSCs thatwere not contacted with the vector and are non-modified HSCs that is notcarrying an exogenous copy of a nucleic acid encoding a PD-L1. In oneembodiment, there is up to ten fold increase in the number of PD-L1⁺expressing cells compared to control HSCs that were not contacted withthe vector and are non-modified HSCs, that is not carrying an exogenouscopy of a nucleic acid encoding a PD-L1.

In one embodiment, the modified HSCs express increased amount of PD-L1.In one embodiment, there is at least one fold increase in the amount ofPD-L1⁺ expressed compared to control HSCs which are HSCs that were notcontacted with the vector and are non-modified HSCs that is not carryingan exogenous copy of a nucleic acid encoding a PD-L1. In one embodiment,there is up to ten fold increase in the amount of PD-L1⁺ expressedcompared to control HSCs that were not contacted with the vector and arenon-modified HSCs that is not carrying an exogenous copy of a nucleicacid encoding a PD-L1.

In one embodiment of the population of PD-L1⁺ expressing HSCs, the exvivo method, or the composition described herein, the PD-L1⁺ expressingHSCs express increased amount of PD-L1. In one embodiment, there is atleast one fold increase in the number of PD-L1⁺ expressing cellscompared to control HSCs which are non-PGE₂ incubated and stimulatedHSCs. In one embodiment, there is up to ten fold increase in the numberof PD-L1⁺ expressing cells compared to control HSCs that are non-PGE₂contacted/incubated and stimulated HSCs.

In one embodiment, the modified HSCs exhibit an increase expression ofPD-L1 over control, non-modified HSCs.

In other embodiments, the increase in the number of PD-L1⁺ expressingcells or the increase in the amount of PD-L1 expressed is at least 1%higher, at least 3% higher, at least 5% higher, at least 8% higher, atleast 10% higher, at least 20% higher, at least 30% higher, at least 40%higher, at least 50% higher, at least 60% higher, at least 70% higher,at least 80% higher, at least 90% higher, at least 1-fold higher, atleast 2-fold higher, at least 5-fold higher, at least 10 fold higher, atleast 100 fold higher, at least 1000-fold higher, or more than acomparable control non-modified HSCs or non-PGE₂ stimulated cells.

Programmed cell death protein 1, also known as PD-1 and cluster ofdifferentiation 279 (CD279), is a receptor protein that in humans isencoded by the PDCD1 gene. PD-1 is a cell surface receptor that belongsto the immunoglobulin superfamily and is expressed on activated T cellsand pro-B cells. PD-1 binds two ligands, PD-L1 (also known as B7 homolog1 (B7-H1) or cluster of differentiation 274 (CD 274)) and PD-L2. The twoligands of PD-1, PD-L1 and PD-L2, are members of the B7 family.

PD-1 and its ligands play an important role in down regulating theimmune system by preventing the activation of T-cells. PD-L 1/PD-1interaction deactivates T cell's cytotoxic activity and leads to theinhibition of immune system. This in turn reduces autoimmunity andpromotes self-tolerance. The inhibitory effect of PD-1 is accomplishedthrough a dual mechanism of promoting apoptosis (programmed cell death)in antigen specific T-cells in lymph nodes while simultaneously reducingapoptosis in regulatory T cells (suppressor T cells).

PD-L1, one of the ligand of the receptor PD-1, is a 40 kDa type 1transmembrane protein encoded by the CD274 gene (Gene ID: 29126). Otherabbreviated symbols for PD-L1 are B7-H, B7H1, PD-L1, PDCD1L1, PDCD1LG1,and PDL1PD-L1. The human CD274 gene can be found on chromosome 9 at thelocation NC 000009.12 (5450381 . . . 5470567) according to the Assemblyfrom the Genome Reference Consortium Human Build 38 patch release 2(GRCh38.p2), under RefSeq or GENBANK assembly accession No:GCF_000001405.28, dated Dec. 5, 2014. The mRNA of the human PD-L1 can befound at GENBANK accession Nos: NM 001267706.1, NM 014143.3, BC113734.1,BC113736.1, BC074984.2 and BC069381.1.

In one embodiment, the mRNA of the human PD-L1 is the isoform bprecursor of the mRNA (variant 2) having the DNA sequence ofatgaggatattt gctgtcttta tattcatgac ctactggcat ttgctgaacg ccccatacaacaaaatcaac caaagaattt tggttgtgga tccagtcacc tctgaacatg aactgacatgtcaggctgag ggctacccca aggccgaagt catctggaca agcagtgacc atcaagtcctgagtggtaag accaccacca ccaattccaa gagagaggag aagcttttca atgtgaccagcacactgaga atcaacacaa caactaatga gattttctac tgcactttta ggagattagatcctgaggaa aaccatacag ctgaattggt catcccagaa ctacctctgg cacatcctccaaatgaaagg actcacttgg taattctggg agccatctta ttatgccttg gtgtagcactgacattcatc ttccgtttaa gaaaagggag aatgatggat gtgaaaaaat gtggcatccaagatacaaac tcaaagaagc aaagtgatac acatttggag gagacgtaa (SEQ. ID. NO: 1).This variant 2 represents the shorter transcript and encodes the shorterisoform b.

In one embodiment, the mRNA of the human PD-L1 is the isoform aprecursor of the mRNA (variant 1) having the DNA sequence ofatgaggatattt gctgtcttta tattcatgac ctactggcat ttgctgaacg catttactgtcacggttccc aaggacctat atgtggtaga gtatggtagc aatatgacaa ttgaatgcaaattcccagta gaaaaacaat tagacctggc tgcactaatt gtctattggg aaatggaggataagaacatt attcaatttg tgcatggaga ggaagacctg aaggttcagc atagtagctacagacagagg gcccggctgt tgaaggacca gctctccctg ggaaatgctg cacttcagatcacagatgtg aaattgcagg atgcaggggt gtaccgctgc atgatcagct atggtggtgccgactacaag cgaattactg tgaaagtcaa tgccccatac aacaaaatca accaaagaattttggttgtg gatccagtca cctctgaaca tgaactgaca tgtcaggctg agggctaccccaaggccgaa gtcatctgga caagcagtga ccatcaagtc ctgagtggta agaccaccaccaccaattcc aagagagagg agaagctttt caatgtgacc agcacactga gaatcaacacaacaactaat gagattttct actgcacttt taggagatta gatcctgagg aaaaccatacagctgaattg gtcatcccag aactacctct ggcacatcct ccaaatgaaa ggactcacttggtaattctg ggagccatct tattatgcct tggtgtagca ctgacattca tcttccgtttaagaaaaggg agaatgatgg atgtgaaaaa atgtggcatc caagatacaa actcaaagaagcaaagtgat acacatttgg aggagacgtaa (SEQ. ID. NO: 2). This variant 1represents the longest transcript and encodes the longer isoform a.

PD-L1 plays a major role in suppressing the immune system duringparticular events such as pregnancy, tissue allografts, autoimmunedisease and other disease states such as hepatitis. Normally the immunesystem reacts to foreign antigens where there is some accumulation inthe lymph nodes or spleen which triggers a proliferation ofantigen-specific CD8+ T cell. The formation of PD-1 receptor/PD-L1 orB7.1 receptor/PD-L1 ligand complex transmits an inhibitory signal whichreduces the proliferation of these CD8+ T cells at the lymph nodes andsupplementary to that PD-1 is also able to control the accumulation offoreign antigen specific T cells in the lymph nodes through apoptosiswhich is further mediated by a lower regulation of the gene BCL-2.

PD-L1 protein is upregulated on macrophages and dendritic cells (DC) inresponse to LPS and GM-CSF treatment, and on T cells and B cells uponTCR and B cell receptor signaling. PD-L1 is expressed in a variety oftissues and cells, e.g., heart, lung, thymus, spleen, kidney and HSCs.PD-L1 is expressed on almost all murine tumor cell lines, including PA1myeloma, P815 mastocytoma, and B16 melanoma upon treatment with IFN-γ.

In one embodiment, the nucleic acid encoding a PD-L1 encodes a humanPD-L1.

In one embodiment of the population of modified HSCs, the ex vivomethod, or the composition described herein, the nucleic acid encodingPD-L1 is a copy DNA (cDNA). In one embodiment, the cDNA encoding PD-L1is an mRNA. In one embodiment, the mRNA is SEQ. ID. NO: 1 or 2. In otherembodiments, the mRNA is derived from the GenBank accession Nos:NM_001267706.1, NM_014143.3, BC113734.1, BC113736.1, BC074984.2 orBC069381.1.

In another embodiment of the population of modified HSCs, the ex vivomethod, or the composition described herein, the nucleic acid encodingPD-L1 is a genomic DNA. In one embodiment, the genomic DNA encodingPD-L1 is derived from the GenBank assembly accession No:GCF_000001405.28.

In one embodiment of the population of modified HSCs, the ex vivomethod, or the composition described herein, the nucleic acid isintegrated into the genome of the HSC cells.

In one embodiment of the population of modified HSCs, the ex vivomethod, or the composition described herein, the nucleic acid isintroduced into the cells via a vector.

In one embodiment of the population of modified HSCs, the ex vivomethod, or the composition described herein, the vector is a viralvector.

In one embodiment of the population of modified HSCs, the ex vivomethod, or the composition described herein, the viral vector is alentiviral vector, an avian virus vector or an adeno-associated virus.

In one aspect of any method, the lentivirus is selected from the groupconsisting of: human immunodeficiency virus type 1 (HIV-1), humanimmunodeficiency virus type 2 (HIV-2), caprine arthritis-encephalitisvirus (CAEV), equine infectious anemia virus (EIAV), felineimmunodeficiency virus (FIV), bovine immune deficiency virus (BIV), andsimian immunodeficiency virus (SIV).

In particular embodiments, cells transduced with the vectorscontemplated herein are genetically modified.

In various embodiments, the genetically modified cells contemplatedherein are transduced in vitro or ex vivo with vectors carrying anexogenous copy of a nucleic acid encoding a PD-L1, and optionallyculture expanded ex vivo. The transduced cells are then administered toa subject in need of gene therapy. Alternatively, the transduced cellscan be cryopreserved prior to administered to a subject in need of genetherapy.

In one embodiment of the population of modified HSCs, the ex vivomethod, or the composition described herein, the cells are mammaliancells.

In one embodiment of the population of modified HSCs, the ex vivomethod, or the composition described herein, the mammalian cells arehuman cells.

HSCs are known to give rise to committed hematopoietic progenitor cells(HPCs) that are capable of generating the entire repertoire of matureblood cells over the lifetime of an organism. The term “hematopoieticstem cell” or “HSC” generally refers to multipotent stem cells that giverise to the all the blood cell types of an organism, including myeloid(e.g., monocytes and macrophages, neutrophils, basophils, eosinophils,erythrocytes, megakaryocytes/platelets, dendritic cells), and lymphoidlineages (e.g., T-cells, B-cells, NK-cells), and others known in the art(See Fei, R., et al., U.S. Pat. No. 5,635,387; McGlave, et al., U.S.Pat. No. 5,460,964; Simmons, P., et al., U.S. Pat. No. 5,677,136;Tsukamoto, et al., U.S. Pat. No. 5,750,397; Schwartz, et al., U.S. Pat.No. 5,759,793; DiGuisto, et al., U.S. Pat. No. 5,681,599; Tsukamoto, etal., U.S. Pat. No. 5,716,827). When transplanted into lethallyirradiated animals or humans, hematopoietic stem and progenitor cellscan repopulate the erythroid, neutrophil-macrophage, megakaryocyte andlymphoid hematopoietic cell pool.

Mature blood cells have a finite lifespan and must be continuouslyreplaced throughout life. Blood cells are produced by the proliferationand differentiation of a very small population of pluripotent HSCs thatalso have the ability to replenish themselves by self-renewal. HSCs aremultipotent, self-renewing progenitor cells that develop from mesodermalhemangioblast cells. HSCs are the blood cells that give rise to all theother blood cells, that includes all the differentiated blood cells fromthe erythroid, lymphoid and myeloid lineages. HSCs are located in theadult bone marrow, peripheral blood, and umbilical cord blood.

During differentiation, the progeny of HSCs progress through variousintermediate maturational stages, generating multi-potentialhematopoietic progenitor cells and lineage-committed hematopoieticprogenitor cells, prior to reaching maturity. Bone marrow (BM) is themajor site of hematopoiesis in humans and, under normal conditions, onlysmall numbers of HSCs and hematopoietic progenitor cells can be found inthe peripheral blood (PB). Treatment with cytokines (in particulargranulocyte colony-stimulating factor; G-CSF), myelosuppressive drugsused in cancer treatment, and compounds that disrupt the interactionbetween hematopoietic cells and BM stromal cells can rapidly mobilizelarge numbers of stem and progenitor cells into the circulation.

“Hematopoietic progenitor cell” as the term is used herein, refers tocells of a hematopoietic stem cell lineage that give rise to all theblood cell types including the myeloid (monocytes and macrophages,neutrophils, basophils, eosinophils, erythrocytes,megakaryocytes/platelets, dendritic cells), and the lymphoid lineages(T-cells, B-cells, NK-cells). A “cell of the erythroid lineage”indicates that the cell being contacted is a cell that undergoeserythropoiesis such that upon final differentiation it forms anerythrocyte or red blood cell (RBC). Such cells belong to one of threelineages, erythroid, lymphoid, and myeloid, originating from bone marrowhematopoietic progenitor cells. Upon exposure to specific growth factorsand other components of the hematopoietic microenvironment,hematopoietic progenitor cells can mature through a series ofintermediate differentiation cellular types, all intermediates of theerythroid lineage, into RBCs. Thus, cells of the “erythroid lineage,” asthe term is used herein, comprise hematopoietic progenitor cells,rubriblasts, prorubricytes, erythroblasts, metarubricytes,reticulocytes, and erythrocytes.

The HSCs, similar to the hematopoietic progenitor cells, are capable ofproliferation and giving rise to more progenitor cells having theability to generate a large number of mother cells that can in turn giverise to differentiated or differentiable daughter cells. The daughtercells themselves can be stimulated to proliferate and produce progenythat subsequently differentiate into one or more mature cell types,while also retaining one or more cells with parental developmentalpotential. The term “stem cell” refers then, to a cell with the capacityor potential, under particular circumstances, to differentiate to a morespecialized or differentiated phenotype, and which retains the capacity,under certain circumstances, to proliferate without substantiallydifferentiating. In one embodiment, the term progenitor or stem cellrefers to a generalized mother cell whose descendants (progeny)specialize, often in different directions, by differentiation, e.g., byacquiring completely individual characters, as occurs in progressivediversification of embryonic cells and tissues. Cellular differentiationis a complex process typically occurring through many cell divisions. Adifferentiated cell may derive from a multipotent cell which itself isderived from a multipotent cell, and so on. While each of thesemultipotent cells may be considered stem cells, the range of cell typeseach can give rise to may vary considerably. Some differentiated cellsalso have the capacity to give rise to cells of greater developmentalpotential. Such capacity may be natural or may be induced artificiallyupon treatment with various factors. In many biological instances, stemcells are also “multipotent” because they can produce progeny of morethan one distinct cell type, but this is not required for “stem-ness.”Self-renewal is the other classical part of the stem cell definition,and it is essential as used in this document. In theory, self-renewalcan occur by either of two major mechanisms. Stem cells may divideasymmetrically, with one daughter retaining the stem state and the otherdaughter expressing some distinct other specific function and phenotype.Alternatively, some of the stem cells in a population can dividesymmetrically into two stems, thus maintaining some stem cells in thepopulation as a whole, while other cells in the population give rise todifferentiated progeny only. Generally, “progenitor cells” have acellular phenotype that is more primitive (i.e., is at an earlier stepalong a developmental pathway or progression than is a fullydifferentiated cell). Often, progenitor cells also have significant orvery high proliferative potential. Progenitor cells can give rise tomultiple distinct differentiated cell types or to a singledifferentiated cell type, depending on the developmental pathway and onthe environment in which the cells develop and differentiate.

Peripheral blood progenitor cells (PBPC) have become the preferredsource of hematopoetic progenitor cells and HSCs for allogeneic andautologous transplantation because of technical ease of collection andshorter time required for engraftment. Traditionally, granulocyte-colonystimulating factor (G-CSF) has been used to stimulate more PBPC andrelease of hematopoetic progenitor cells from the bone marrow. Althoughregimens using G-CSF usually succeed in collecting adequate numbers ofPBPC from healthy donors, 5%40% will mobilize stem cells poorly and mayrequire multiple large volume apheresis or bone marrow harvesting.

In one embodiment of the population of modified HSCs, the ex vivomethod, or the composition described herein, prior to the modification,the sample of HSCs is obtained from the bone marrow, umbilical cord,chorionic villi, amniotic fluid, placental blood, cord blood orperipheral blood. In one embodiment, the HSCs are isolated from the bonemarrow, umbilical cord, chorionic villi, amniotic fluid, placentalblood, cord blood or peripheral blood.

In one embodiment of the population of modified HSCs, the ex vivomethod, or the composition described herein, the sample of HSCs isobtained from mobilized peripheral blood. Methods of mobilizing HSCsfrom the places of origin or storage are known in the art. For example,treatment with cytokines, in particular granulocyte colony-stimulatingfactor (G-CSF) and compounds (e.g., plerixafor, a chemokine CXCR4antagonist) that disrupt the interaction between HSCs and bone marrow(BM) stromal cells can rapidly mobilize large numbers of hematopoieticstem and hematopoietic progenitor cells into the circulation. In oneembodiment of the population of modified HSCs, the ex vivo method, orthe composition described herein, the sample of HSCs is CD34⁺ selectedcells obtained from the bone marrow, umbilical cord, chorionic villi,amniotic fluid, placental blood, cord blood or peripheral blood, ormobilized peripheral blood.

In one embodiment of the population of modified HSCs, the ex vivomethod, or the composition described herein, the HSCs are CD34⁺ cells.In other embodiments, the HSCs are CD38⁺ cells. In other embodiments,the HSCs are c-kit⁺ cells.

In one embodiment of the population of modified HSCs, the ex vivomethod, or the composition described herein, the HSCs are hematopoieticprogenitor cells. In one embodiment, these hematopoietic progenitorcells are CD34⁺ cells. In other embodiments, these hematopoieticprogenitor cells are CD38^(lo/−) cells. In other embodiments, thesehematopoietic progenitor cells are c-kit⁺ cells.

In one embodiment of the population of modified HSCs, the ex vivomethod, or the composition described herein, the HSCs are erythroidprogenitor cells. In one embodiment, these erythroid progenitor cellsare CD34⁺ cells.

In one embodiment of the population of modified HSCs, the ex vivomethod, or the composition described herein, the HSCs are erythroidcells. In one embodiment, these erythroid cells are CD34⁺ cells.

In one embodiment of the population of modified HSCs, the ex vivomethod, or the composition described herein, the HSC is selected for theCD34⁺ surface marker prior to the contacting with the vector carryingthe exogenous copy of the nucleic acid described herein.

In other embodiment of the population of modified HSCs, the ex vivomethod, or the composition described herein, the HSC is selected for theCD38^(lo/−) surface marker prior to the contacting with the vectorcarrying the exogenous copy of the nucleic acid described herein.

In one embodiment of the population of modified HSCs, the ex vivomethod, or the composition described herein, the HSC is selected for thec-kit⁺ surface marker prior to the contacting with the vector carryingthe exogenous copy of the nucleic acid described herein. Positive ornegative selection for the described surface markers can be performed byany method known in the art, e.g., using the anti-CD34 immunomagneticbead described in the Example section.

It one embodiment of the population of modified HSCs, the ex vivomethod, or the composition described herein, the isolated CD34⁺ HSC iscontacted with the PGE₂ composition described herein or contacted withthe vector carrying the exogenous copy of the nucleic acid describedherein.

In one embodiment of the population of modified HSCs, the ex vivomethod, or the composition described herein, the HSC has at least one ofthe cell surface marker characteristic of HSCs: CD34⁺, CD59⁺,Thy1/CD90⁺, CD38^(lo/−) and C-kit/CD117⁺. Preferably, the HSCs haveseveral of these markers.

In one embodiment of the population of modified HSCs, the ex vivomethod, or the composition described herein, the HSCs are CD34⁺, CD59⁺,Thy1/CD90⁺, CD38^(lo/−), and C-kit/CD117⁺.

In one embodiment of the population of modified HSCs, the ex vivomethod, or the composition described herein, the HSCs are CD 133⁺.

In one embodiment of the population of modified HSCs, the ex vivomethod, or the composition described herein, the hematopoieticprogenitor cells are CD 133⁺.

In one embodiment of the population of modified HSCs, the ex vivomethod, or the composition described herein, the hematopoieticprogenitor cells of the erythroid lineage have the cell surface markercharacteristic of the erythroid lineage: CD71 and Ter119.

In one embodiment of the population of modified HSCs, the ex vivomethod, or the composition described herein, the HSCs have the cellsurface marker characteristic of the erythroid lineage: CD71 and Ter119.

In one embodiment of the population of modified HSCs, the ex vivomethod, or the composition described herein, the HSCs have at least oneof the cell surface marker selected from the group consisting of CD34⁺,CD59⁺, Thy1/CD90⁺, CD38^(lo/−), and C-kit/CD117⁺.

In one embodiment of the population of modified HSCs, the ex vivomethod, or the composition described herein, the HSCs are positivelyselected for at least one of the cell surface marker selected from thegroup consisting of CD34⁺, CD59⁺, Thy1/CD90⁺, and C-kit/CD117⁺. Inanother embodiment of the population of modified HSCs, the ex vivomethod, or the composition described herein, the HSCs are negativelyselected for CD38^(lo/−).

In one embodiment of the population of modified HSCs, the ex vivomethod, or the composition described herein, the sample of HSCs isobtained from a healthy individual or subject.

In one embodiment of the population of modified HSCs, the ex vivomethod, or the composition described herein, the HSCs are obtained orisolated from an individual with a diagnosed disease or disorder or anindividual who is an organ or bone marrow transplant recipient.

In one embodiment of the population of modified HSCs, the ex vivomethod, or the composition described herein, the HSCs are obtained orisolated from an individual who is newly diagnosed with T1D.

In one embodiment of the population of modified HSCs, the ex vivomethod, or the composition described herein, the diagnosed disease ordisorder is an autoimmune disease or disorder.

In one embodiment of the population of modified HSCs, the ex vivomethod, or the composition described herein, the autoimmune disease ordisorder is T1D.

In one embodiment of the population of modified HSCs, the ex vivomethod, or the composition described herein, the contacting of the HSCswith the vector carrying the exogenous copy of the nucleic aciddescribed herein is repeated at least once. That is, after the initialfirst contacting of the HSC with the virus or vector described herein,the cell is washed and collected, and the washed cell is then contactedfor a second time with the virus or vector carrying a nucleic acidmolecule described herein. These cells are then washed a second time andcollected.

In other embodiments of the population of modified HSCs, the ex vivomethod, or the composition described herein, the contacting is repeatedat least twice after the initial first contacting.

In one embodiment of the population of modified HSCs, the ex vivomethod, or the composition described herein, the isolated or collectedHSCs are ex vivo cultured before and/or after the introduction of theexogenous copy of a nucleic acid encoding a PD-L1. In one embodiment,the ex vivo culturing serve to expand or grow the population of presentcells, that is, to increase the number of similar cells.

In one embodiment of the population of modified HSCs, the ex vivomethod, or the composition described herein, the isolated or collectedHSCs are ex vivo cultured before contacting, incubation or stimulationwith PGE₂.

In one embodiment of the population of modified HSCs, the ex vivomethod, or the composition described herein, the isolated or collectedHSCs are ex vivo cultured after contacting, incubation or stimulationwith PGE₂.

In one embodiment of the population of modified HSCs, the ex vivomethod, or the composition described herein, the isolated or collectedHSCs are ex vivo cultured before and after contacting, incubation orstimulation with PGE₂.

In another embodiment, the ex vivo culture expansion take place prior touse, for example, use in cryopreservation, or use inimplantation/engraftment into a recipient subject.

In one embodiment of the population of modified HSCs, the ex vivomethod, or the composition described herein, the HSCs are cryopreservedprior to the introduction of the exogenous copy of a nucleic acidencoding a PD-L1.

In one embodiment of the population of modified HSCs, the ex vivomethod, or the composition described herein, the HSCs are cryopreservedafter the introduction of the exogenous copy of a nucleic acid encodinga PD-L1.

In one embodiment of the population of modified HSCs, the ex vivomethod, or the composition described herein, the HSCs are cryopreservedprior to and after the introduction of the exogenous copy of a nucleicacid encoding a PD-L1.

In one embodiment of the population of modified HSCs, the ex vivomethod, or the composition described herein, the HSCs are cryopreservedprior to contacting, incubation or stimulation with PGE₂, or aftercontacting, incubation or stimulation with PGE₂, or both prior to andafter contacting, incubation or stimulation with PGE₂.

In one embodiment of the population of modified HSCs, the ex vivomethod, or the composition described herein, the modified PD-L1⁺expressing HSCs are ex vivo culture expanded and then cryopreservedprior to use. For example, ex vivo cell expansion and/orimplantation/engraftment into a subject.

The cells described herein can be cryopreserved by any methods known inthe art. As used herein, “cryopreserving” refers to the preservation ofcells by cooling to low sub-zero temperatures, such as (typically) 77 Kor −196° C. (the boiling point of liquid nitrogen). Cryopreservationalso refers to preserving cells at a temperature between 4-10° C. Atthese low temperatures, any biological activity, including thebiochemical reactions that would lead to cell death, is effectivelystopped. Cryoprotective agents are often used at sub-zero temperaturesto prevent the cells being preserved from damage due to freezing at lowtemperatures or warming to room temperature.

Freezing is destructive to most living cells. Upon cooling, as theexternal medium freezes, cells equilibrate by losing water, thusincreasing intracellular solute concentration. Below about 10°-15° C.,intracellular freezing will occur. Both intracellular freezing andsolution effects are responsible for cell injury (Mazur, P., 1970,Science 168:939-949). It has been proposed that freezing destructionfrom extracellular ice is essentially a plasma membrane injury resultingfrom osmotic dehydration of the cell (Meryman, H. T., et al., 1977,Cryobiology 14:287-302).

Cryoprotective agents and optimal cooling rates can protect against cellinjury. Cryoprotective agents which can be used include but are notlimited to dimethyl sulfoxide (DMSO) (Lovelock, J. E. and Bishop, M. W.H., 1959, Nature 183:1394-1395; Ashwood-Smith, M. J., 1961, Nature190:1204-1205), glycerol, polyvinylpyrrolidine (Rinfret, A. P., 1960,Ann. N.Y. Acad. Sci. 85:576), Dextran, trehalose, CryoSoFree (SignaAldrich Co.) and polyethylene glycol (Sloviter, H. A. and Ravdin, R. G.,1962, Nature 196:548). The preferred cooling rate is 1° to 3° C./minute.After at least two hours, the T-cells have reached a temperature of −80°C. and can be placed directly into liquid nitrogen (−196° C.) forpermanent storage such as in a long-term cryogenic storage vessel.

In one embodiment of the population of modified HSCs, the ex vivomethod, or the composition described herein, the PGE₂ is 16,16-Dimethylprostaglandin E2 (dmPGE₂).

Uses of PD-L1 Expressing HSCs and Compositions Comprising these HSCs

The modified PD-L1 expressing HSCs described herein can be used to treatan autoimmune disorder, getting to the root cause of an autoimmunedisorder, a defect in immunoregulation. The modified PD-L1 expressingHSCs are used to modulate or suppress an immune response in a subjecthaving the autoimmune disorder. In one embodiment, the autoimmunedisorder is T1D. Subjects with T1D have defects in producing PD-L1expression HSCs. The modified PD-L1 expressing HSCs are used tosupplement this defect and modulate or suppress the immune responseagainst the 13 islet cells of the pancreas of the subject having T1D. Inone embodiment, the modified PD-L1 expressing HSCs described herein areused to treat T1D in a subject diagnosed with T1D. In one embodiment,the subject is newly diagnosed with T1D. As used herein, the term “newlydiagnosed” refers to diagnosis for the disorder for less than onecalendar year. In one embodiment, the subject is newly been detected tohave self-autoantibodies associated with T1D, e.g., GAD65 autoantibody,and islet antigen 2 autoantibody. As used herein, the term “newlydetected” refers to the detection of self-autoantibodies associated withT1D in the last 6 calendar months.

For example, a human subject has been newly diagnosed with T1D. A sampleof HSCs can be harvested from this subject. The HSCs obtained can be exvivo expanded to increase the number of available HSCs for theprocedures described herein to increase PD-L1 expression. A sample ofHSCs can be transfected with an exogenous of a PD-L1 cDNA to bring aboutoverexpression of PD-L1 in the transfected HSCs. Alternatively, a sampleof HSCs can be contacted ex vivo with PGE₂ as described herein tostimulate increased PD-L1 expression in the PGE₂-contacted HSCs. BothPGE₂ and steroids such as dexamethasome also can be use together tostimulate PD-L1 expression. Either method of increasing PD-L1 expressionand increasing the pool of PD-L1 expressing HSCs can be used. Theresultant HSCs are then analysed to confirmed increased PD-L1 expressioncompared to non-transfected HSCs or non-PGE₂-contacted HSCsrespectively. The resultant HSCs can be further ex vivo expanded toincrease the number of available HSCs for transplantation back into thesubject. The resultant HSCs can also be ex vivo expanded to increase thenumber of available PD-L1 expressing HSCs for cryopreservation and fortransplantation back into the subject, i.e., have a portion of PD-L1expressing HSCs kept in cryostorage and another portion fortransplantation back into the subject. The PD-L1 expressing HSCs areautologous to the recipient subject because the original HSCs wereobtained from the same subject, therefore the HSCs are HLA matched tothe subject.

For example, a human subject has been newly been detected to haveself-autoantibodies associated with T1D, e.g., GAD65 autoantibody, andislet antigen 2 autoantibody. The four autoantibodies that are markersof beta cell autoimmunity in type 1 diabetes are: islet cell antibodies(ICA, against cytoplasmic proteins in the beta cell), antibodies toglutamic acid decarboxylase (GAD-65), insulin autoantibodies (IAA), andIA-2A, to protein tyrosine phosphatase. Autoantibodies against GAD 65are found in 80% of type 1 diabetics at clinical presentation. Presenceof ICA and IA-2A at diagnosis for type 1 diabetes range from 69-90% and54-75%, respectively. IAA prevalence correlates inversely with age atonset of diabetes; it is usually the first marker in young children atrisk for diabetes and found in approximately 70% of young children attime of diagnosis. The subject is not yet symptomatic for T1D (ie.,hyperglycemia). The therapeutic methods using the PD-L1 expressing HSCsare used to delay onset of hyperglycemia for such an individual.Hyperglycemia, or high blood sugar is a condition in which an excessiveamount of glucose circulates in the blood plasma. This is generally ablood sugar level higher than 11.1 mmol/l (200 mg/dl), but symptoms maynot start to become noticeable until even higher values such as 15-20mmol/l (˜250-300 mg/dl). A subject with a consistent range between ˜5.6and ˜7 mmol/1 (100-126 mg/dl) (American Diabetes Association guidelines)is considered hyperglycemic, while above 7 mmol/l (126 mg/dl) isgenerally held to have diabetes. In one embodiment, the subject hasblood sugar below 11.1 mmol/l (200 mg/dl). In another embodiment, theblood sugar below 15 mmol/l (˜250 mg/dl) or below 20 mmol/1300 mg/dl).Administering the PD-L1⁺ cells can delay the onset of diabetes.

The modified PD-L1 expressing HSCs described herein can also be used tosuppress an immune response in a subject who is an organ or bone marrowtransplant recipient, or a subject who is going to be recipient in thenear future. The modified PD-L1 expressing HSCs are used to prevent ortreat or both prevent and treat host-versus-graft disease (GVHD). GvHDis a medical complication following the receipt of transplanted tissuefrom a genetically different person. GvHD is commonly associated withstem cell or bone marrow transplant but the term also applies to otherforms of tissue graft. A sample of HSCs can be harvested from thissubject. The HSCs obtained can be ex vivo expanded to increase thenumber of available HSCs for the procedures described herein to increasePD-L1 expression. A sample of HSCs can be transfected with an exogenousof a PD-L1 cDNA to bring about overexpression of PD-L1 in thetransfected HSCs. Alternatively, a sample of HSCs can be contacted exvivo with PGE₂ as described herein to stimulate increase PD-L1 in thePGE₂-contacted HSCs. Both PGE₂ and a steroid such as dexamethasome canbe use together to stimulate PD-L1 expression. Either method ofincreasing PD-L1 expression and increasing the pool of PD-L1 expressingHSCs can be used. The resultant HSCs are then analysed to confirmedincreased PD-L1 expression compared to non-transfected HSCs ornon-PGE₂-contacted HSCs respectively. The resultant HSCs can be furtherex vivo expanded to increase the number of available HSCs fortransplantation back into the subject. The resultant HSCs can also be exvivo expanded to increase the number of available PD-L1 expressing HSCsfor cryopreservation and for transplantation back into the subject.

Accordingly, in one embodiment, provided herein is a compositioncomprising the PD-L1 expressing hematopoietic stem cells describedherein or PD-L1⁺ HSCs produced by any one of the method described hereinfor use in the prevention or treatment of an autoimmune disease ordisorder, for use in suppressing an immune response in a subject, foruse in the delay of the onset of T1D in a subject at risk of developingT1D, for use in the prevention and delay of an allogenic tissue or organtransplant rejection, and for the treatment of T1D in adult andpediatric subjects.

In one embodiment, provided herein is a composition comprising the PD-L1expressing HSCs described herein or PD-L1⁺ HSCs produced by any one ofthe method described herein for the manufacture of medicament for use inthe prevention or treatment of an autoimmune disease or disorder, in thesuppression of an immune response in a subject, in the delay of theonset of T1D in a subject at risk of developing T1D, in the preventionand delay of an allogenic tissue or organ transplant rejection, and forthe treatment of T1D in adult and pediatric subjects.

In another embodiment, provided herein is a population of PD-L1expressing HSCs described herein or PD-L1⁺ HSCs produced by any one ofthe method described herein for use in the prevention or treatment of anautoimmune disease or disorder, for use in suppressing an immuneresponse in a subject, for use in the delay of the onset of T1D in asubject at risk of developing T1D, for use in the prevention and delayof an allogenic tissue or organ transplant rejection, and for thetreatment of T1D in adult and pediatric subjects.

In another embodiment, provided herein is a population of PD-L1expressing HSCs described herein or PD-L1⁺ HSCs produced by any one ofthe method described herein for the manufacture of medicament for use inthe prevention or treatment of an autoimmune disease or disorder, in thesuppression of an immune response in a subject, in the delay of theonset of T1D in a subject at risk of developing T1D, in the preventionand delay of an allogenic tissue or organ transplant rejection, and forthe treatment of T1D in adult and pediatric subjects.

Accordingly, in one embodiment, provided herein is a method of treatingan autoimmune disorder or suppressing an immune response in a subject inneed thereof, the method comprising administering to a subject acomposition comprising a population of modified HSCs described herein.In one embodiment, the modified HSCs express PD-L1. In one embodiment,the modified HSCs exhibit increase expression of PD-L1 over the control,non-modified HSCs. In one embodiment, the method further comprisesidentifying a subject afflicted with an autoimmune disease or disorder.In another embodiment, the method further comprises selecting a subjecthaving an autoimmune disease or disorder, or an individual who is anorgan or bone marrow transplant recipient.

In one embodiment, provided herein is a method of preventinghost-versus-graft disease, or organ or tissue graft rejection in asubject in need thereof, the method comprising administering to asubject a composition comprising a population of modified HSCs describedherein. In one embodiment, the subject has received an allogenic tissueor organ graft. In one embodiment, the modified HSCs carry an exogenouscopy of a nucleic acid encoding a PD-L1. In one embodiment, the modifiedHSCs express PD-L1. In one embodiment, the modified HSCs exhibitincrease expression of PD-L1 over the control, non-modified HSCs.

In one embodiment, the modified HSCs are PGE₂-stimulated, PD-L1⁺expressing HSCs described herein. In one embodiment, the modified HSCsare PGE₂ and dexamethasone-stimulated, PD-L1⁺ expressing HSCs describedherein.

In one embodiment, provided herein is a method of delaying the onset ofType 1 diabetes in a subject in need thereof, the method comprisingadministering to a subject a composition comprising a population ofmodified HSCs described herein. In one embodiment, the subject is newlybeen noted to have detectable amounts of a self-autoantibody associatedwith T1D. In one embodiment, the subject does not have clinicalhyperglycemia. In one embodiment, the subject is a pediatric patientunder the age of 20 years old. In other embodiments, the subject is apediatric patient under the age of 15 years old, 10 years old, 5 yearsold, and 1 years old. In one embodiment, the modified HSCs carry anexogenous copy of a nucleic acid encoding a PD-L1. In one embodiment,the modified HSCs express PD-L1. In one embodiment, the modified HSCsexhibit increase expression of PD-L1 over the control, non-modifiedHSCs.

In one embodiment, the modified HSCs are PGE₂-stimulated, PD-L1⁺expressing HSCs described herein. In one embodiment, the modified HSCsare PGE₂ and dexamethasone-stimulated, PD-L1⁺ expressing HSCs describedherein.

A variety of autoimmune diseases or disorders are known in the art, forexample, those described in the definition section. The skilledphysician would be able to diagnose an autoimmune disease or disorderthat is known in the art.

In another embodiment, the method further comprises selecting a subjectin need of immune response suppression. In general, deliberately inducedimmunosuppression is performed to prevent the body from rejecting anorgan transplant or an allograft transplant, treating GVHD after anorgan or bone marrow transplant, or for the treatment of autoimmunediseases such as systemic lupus erythematosus, rheumatoid arthritis orCrohn's disease. In some embodiments, an organ transplantation includeliver, skin, lung transplantation, pancreas, kidney, ovary, colon,intestine, and heart transplantation.

In one embodiment, provided herein is a method of treating an autoimmunedisorder or suppressing an immune response in a subject in need thereof,the method comprising administering to a subject a compositioncomprising a population of modified HSCs where the modified HSCs carryan exogenous copy of a nucleic acid encoding a PD-L1. In one embodiment,the modified HSCs express PD-L1. In one embodiment, the modified HSCsexhibit increase expression of PD-L1 over the control, non-modifiedHSCs.

In one embodiment, provided herein is a method of treating an autoimmunedisorder or suppressing an immune response in a subject in need thereof,the method comprising administering to a subject a compositioncomprising a population of modified PD-L1⁺ expressing HSCs where themodified HSCs are produced by an ex vivo method comprising contacting asample of HSCs with a vector carrying an exogenous copy of a nucleicacid encoding a PD-L1 to modify the HSCs whereby the exogenous copy of anucleic acid is introduced into the HSCs, thereby producing a populationof modified HSCs cells expressing PD-L1. In one embodiment, the methodfurther comprises establishing the expression of PD-L1 on the resultantmodified HSCs.

In another embodiment, the method further comprises ex vivo culturingthe resultant modified cells after contact with the vector. In anotherembodiment, the method further comprises ex vivo culturing the resultantmodified cells after establishing the expression of PD-L1 on theresultant modified HSCs. In another embodiment, the method furthercomprises ex vivo culturing the resultant modified cells after contactwith the vector and ex vivo culturing the resultant modified cells afterestablishing the expression of PD-L1 on the resultant modified HSCs.

In one embodiment, provided herein is a method of treating an autoimmunedisorder or suppressing an immune response in a subject in need thereof,the method comprising administering to a subject a compositioncomprising a population of PGE₂-stimulated, PD-L1+ expressing HSCsdescribed herein. In one embodiment, the method further comprisesidentifying a subject afflicted with an autoimmune disease or disorder.

In another embodiment, the method further comprises selecting a subjecthaving an autoimmune disease or disorder, or a subject who is an organor bone marrow transplant recipient, or a subject who is an organ orbone marrow transplant recipient and is at risk of developing GVHD. Forexample, a subject who has received an allogenic graft transplant. Inanother embodiment, the method further comprises selecting a subject inneed of immune response suppression. For example, a subject who an organor bone marrow transplant recipient and is at risk of developing GVHD.

In one embodiment, provided herein is a method of treating an autoimmunedisorder or suppressing an immune response in a subject in need thereof,the method comprising administering to a subject a compositioncomprising a population of PD-L1⁺ expressing HSCs wherein the HSCs arestimulated to express PD-L1⁺ by contacting with PGE₂. In one embodiment,the stimulated HSCs exhibit an increase expression of PD-L1 over thecontrol, non-PGE₂-stimulated HSCs.

In one embodiment, provided herein is a method of treating an autoimmunedisorder or suppressing an immune response in a subject in need thereof,the method comprising administering to a subject a compositioncomprising a population of PD-L1⁺ expressing HSCs where the PD-L1⁺expressing HSCs are produced by an ex vivo method comprising contactinga sample of HSCs with PGE₂ at 10 μM concentration for about 60 min at37° C., thereby producing a population of PGE₂-stimulated HSCs cellsexpressing PD-L1.

In one embodiment, provided herein is a method of treating an autoimmunedisorder or suppressing an immune response in a subject in need thereof,the method comprising administering to a subject a compositioncomprising a population of PD-L1⁺ expressing HSCs where the PD-L1⁺expressing HSCs are produced by an ex vivo method comprising contactinga sample of HSCs with PGE₂ at 0.1 μM concentration for at least 24 hrsat 37° C., thereby producing a population of PGE₂-stimulated HSCs cellsexpressing PD-L1.

In one embodiment of the above described method, the method furthercomprises washing the contacted cells to remove excess PGE₂. In oneembodiment, the method further comprises establishing the expression ofPD-L1 on the PGE₂-stimulated HSCs. In another embodiment, the methodfurther comprises ex vivo culturing of the sample of HSCs prior toPGE₂-stimulation. This ex vivo culturing expands the number of cellsavailable for PGE₂-stimulation. In another embodiment, the methodfurther comprises ex vivo culturing of the PGE₂-stimulated HSCs aftercontact with PGE₂, or ex vivo culturing of the PGE₂-stimulated HSCsafter establishing the expression of PD-L1 on the PGE₂-stimulated HSCs,or both ex vivo culturing of the PGE₂-stimulated HSCs after contact withPGE₂ and ex vivo culturing of the PGE₂-stimulated HSCs afterestablishing the expression of PD-L1 on the PGE₂-stimulated HSCs. Thisex vivo culturing expands the number of PD-L1 expressing cells availablefor therapy.

In one embodiment, provided herein is a method of treating an autoimmunedisorder or suppressing an immune response in a subject in need thereof,the method comprising providing a population of HSCs; contacting thesample of HSCs with a vector carrying an exogenous copy of a nucleicacid encoding a PD-L1 to produce a population of modified HSCs cellsexpressing PD-L1; and administering the population of modified, PD-L1⁺expressing HSCs into a recipient subject to promote immunoregulation andimmune self-tolerance in the recipient subject. In one embodiment, themethod further comprises establishing the expression of PD-L1 on theresultant modified HSCs. In another embodiment, the method furthercomprises ex vivo culturing the resultant modified cells after contactwith the vector and/or ex vivo culturing the resultant modified cellsafter establishing the expression of PD-L1 on the resultant modifiedHSCs. In one embodiment, the treatment method further comprisesidentifying a recipient subject afflicted with an autoimmune disease ordisorder and is in need of increased immunoregulation and immuneself-tolerance. In another embodiment, the treatment method furthercomprises selecting a recipient subject having an autoimmune disease ordisorder or is in need of suppressing an immune response. In anotherembodiment, the treatment method further comprises identifying andselecting a donor subject to provide the sample of HSCs for contactingwith the described vector or stimulation with PGE₂. In one embodiment,the donor subject and recipient subject are the same subject, that isthe recipient subject would be administered autologous HSCs. In anotherembodiment, the donor subject and recipient subject are differentsubjects. In another embodiment, the donor subject and recipient subjectat the minimum HLA type matched.

In one embodiment, provided herein is a method of treating an autoimmunedisorder or suppressing an immune response in a subject in need thereof,the method comprising providing a population of HSCs; contacting sampleof HSCs with PGE₂ at 10 μM concentration for about 60 min at 37° C. toproduce a population of PGE₂-stimulated HSCs cells expressing PD-L1; andadministering the population of PGE₂-stimulated PD-L1⁺ expressing HSCsinto a recipient subject to promote immunoregulation and immuneself-tolerance in the recipient subject. In one embodiment, the methodfurther comprises establishing the expression of PD-L1 on the resultantPGE₂-stimulated HSCs.

In one embodiment, provided herein is a method of treating an autoimmunedisorder or suppressing an immune response in a subject in need thereof,the method comprising providing a population of HSCs; contacting sampleof HSCs with PGE₂ at 0.1 μM concentration for at least 24 hrs at 37° C.to produce a population of PGE₂-stimulated HSCs cells expressing PD-L1;and administering the population of PGE₂-stimulated PD-L1⁺ expressingHSCs into a recipient subject to promote immunoregulation and immuneself-tolerance in the recipient subject. In one embodiment, the methodfurther comprises establishing the expression of PD-L1 on the resultantPGE₂-stimulated HSCs.

In another embodiment of the above described methods, thePGE₂-stimulated HSCs are also contacted with steroids such asdexamethasone.

In another embodiment of the above described methods, the method furthercomprises ex vivo culturing the resultant PGE₂-stimulated cells aftercontact with PGE₂, or ex vivo culturing the resultant PGE₂-stimulatedcells after establishing the expression of PD-L1 on the resultantPGE₂-stimulated HSCs, or both ex vivo culturing the resultantPGE₂-stimulated cells after contact with PGE₂ and ex vivo culturing theresultant PGE₂-stimulated cells after establishing the expression ofPD-L1 on the resultant PGE₂-stimulated HSCs.

In one embodiment, the method further comprises identifying a recipientsubject afflicted with an autoimmune disease or disorder and is in needof increased immunoregulation and immune self-tolerance. In oneembodiment, the method further comprises identifying a recipient subjectwho is an organ or bone marrow transplant recipient, and is in need ofincreased immunoregulation and immune self-tolerance. In anotherembodiment, the method further comprises selecting a recipient subjecthaving an autoimmune disease or disorder or who is an organ or bonemarrow transplant recipient. In another embodiment, the treatment methodfurther comprises identifying and selecting a donor subject to providethe sample of HSCs for contacting with PGE₂. In one embodiment, thedonor subject and recipient subject are the same subject, that is therecipient subject would be administered autologous HSCs. In anotherembodiment, the donor subject and recipient subject are differentsubjects. In another embodiment, the donor subject and recipient subjectat the minimum HLA type matched.

In one embodiment, the HSCs are isolated from a host subject,transfected with a vector, cultured (optional), and transplanted backinto the same host, i.e. an autologous cell transplant. In anotherembodiment, the HSCs are isolated from a donor who is an HLA-type matchwith a host (recipient) who is diagnosed with an autoimmune disease ordisorder, or T1D. Donor-recipient antigen type-matching is well known inthe art. The HLA-types include HLA-A, HLA-B, HLA-C, and HLA-D. Theserepresent the minimum number of cell surface antigen matching requiredfor transplantation. That is the transfected cells are transplanted intoa different host, i.e., allogeneic to the recipient host subject. Thedonor's or subject's HSCs can be transfected with a vector or nucleicacid comprising the nucleic acid molecule described herein, thetransfected cells are culture expanded ex vivo, and then transplantedinto the host subject. In one embodiment, the transplanted cellsengrafts in the host subject. The transfected HSCs can also becryopreserved after transfected and stored, or cryopreserved after cellexpansion and stored.

In one embodiment of any one of the method described, the autoimmunedisorder is selected from the group consisting of thyroiditis, type 1diabetes mellitus, Hashimoto's thyroiditis, Graves' disease, celiacdisease, multiple sclerosis, Guillain-Barre syndrome, Addison's disease,and Raynaud's phenomenon, Goodpasture's disease, arthritis (rheumatoidarthritis such as acute arthritis, chronic rheumatoid arthritis, gout orgouty arthritis, acute gouty arthritis, acute immunological arthritis,chronic inflammatory arthritis, degenerative arthritis, type IIcollagen-induced arthritis, infectious arthritis, Lyme arthritis,proliferative arthritis, psoriatic arthritis, Still's disease, vertebralarthritis, and juvenile-onset rheumatoid arthritis, arthritis chronicaprogrediente, arthritis deformans, polyarthritis chronica primaria,reactive arthritis, and ankylosing spondylitis), inflammatoryhyperproliferative skin diseases, psoriasis such as plaque psoriasis,gutatte psoriasis, pustular psoriasis, and psoriasis of the nails, atopyincluding atopic diseases such as hay fever and Job's syndrome,dermatitis including contact dermatitis, chronic contact dermatitis,exfoliative dermatitis, allergic dermatitis, allergic contactdermatitis, dermatitis herpetiformis, nummular dermatitis, seborrheicdermatitis, non-specific dermatitis, primary irritant contactdermatitis, and atopic dermatitis, x-linked hyper IgM syndrome, allergicintraocular inflammatory diseases, urticaria such as chronic allergicurticaria and chronic idiopathic urticaria, including chronic autoimmuneurticaria, myositis, polymyositis/dermatomyositis, juveniledermatomyositis, toxic epidermal necrolysis, scleroderma (includingsystemic scleroderma), sclerosis such as systemic sclerosis, multiplesclerosis (MS) such as spino-optical MS, primary progressive MS (PPMS),and relapsing remitting MS (RRMS), progressive systemic sclerosis,atherosclerosis, arteriosclerosis, sclerosis disseminata, ataxicsclerosis, neuromyelitis optica (NMO), inflammatory bowel disease (IBD)(for example, Crohn's disease, autoimmune-mediated gastrointestinaldiseases, colitis such as ulcerative colitis, colitis ulcerosa,microscopic colitis, collagenous colitis, colitis polyposa, necrotizingenterocolitis, and transmural colitis, and autoimmune inflammatory boweldisease), bowel inflammation, pyoderma gangrenosum, erythema nodosum,primary sclerosing cholangitis, respiratory distress syndrome, includingadult or acute respiratory distress syndrome (ARDS), meningitis,inflammation of all or part of the uvea, iritis, choroiditis, anautoimmune hematological disorder, rheumatoid spondylitis, rheumatoidsynovitis, hereditary angioedema, cranial nerve damage as in meningitis,herpes gestationis, pemphigoid gestationis, pruritis scroti, autoimmunepremature ovarian failure, sudden hearing loss due to an autoimmunecondition, IgE-mediated diseases such as anaphylaxis and allergic andatopic rhinitis, encephalitis such as Rasmussen's encephalitis andlimbic and/or brainstem encephalitis, uveitis, such as anterior uveitis,acute anterior uveitis, granulomatous uveitis, nongranulomatous uveitis,phacoantigenic uveitis, posterior uveitis, or autoimmune uveitis,glomerulonephritis (GN) with and without nephrotic syndrome such aschronic or acute glomerulonephritis such as primary GN, immune-mediatedGN, membranous GN (membranous nephropathy), idiopathic membranous GN oridiopathic membranous nephropathy, membrano- or membranous proliferativeGN (MPGN), including Type I and Type II, and rapidly progressive GN,proliferative nephritis, autoimmune polyglandular endocrine failure,balanitis including balanitis circumscripta plasmacellularis,balanoposthitis, erythema annulare centrifugum, erythema dyschromicumperstans, eythema multiform, granuloma annulare, lichen nitidus, lichensclerosus et atrophicus, lichen simplex chronicus, lichen spinulosus,lichen planus, lamellar ichthyosis, epidermolytic hyperkeratosis,premalignant keratosis, pyoderma gangrenosum, allergic conditions andresponses, allergic reaction, eczema including allergic or atopiceczema, asteatotic eczema, dyshidrotic eczema, and vesicularpalmoplantar eczema, asthma such as asthma bronchiale, bronchial asthma,and auto-immune asthma, conditions involving infiltration of T cells andchronic inflammatory responses, immune reactions against foreignantigens such as fetal A-B-O blood groups during pregnancy, chronicpulmonary inflammatory disease, autoimmune myocarditis, leukocyteadhesion deficiency, lupus, including lupus nephritis, lupus cerebritis,pediatric lupus, non-renal lupus, extra-renal lupus, discoid lupus anddiscoid lupus erythematosus, alopecia lupus, systemic lupuserythematosus (SLE) such as cutaneous SLE or subacute cutaneous SLE,neonatal lupus syndrome (NLE), and lupus erythematosus disseminatus,juvenile onset (Type I) diabetes mellitus, including pediatricinsulin-dependent diabetes mellitus (IDDM), adult onset diabetesmellitus (Type II diabetes), autoimmune diabetes, idiopathic diabetesinsipidus, diabetic retinopathy, diabetic nephropathy, diabeticlarge-artery disorder, immune responses associated with acute anddelayed hypersensitivity mediated by cytokines and T-lymphocytes,sarcoidosis, granulomatosis including lymphomatoid granulomatosis,Wegener's granulomatosis, agranulocytosis, vasculitides, includingvasculitis, large-vessel vasculitis (including polymyalgia rheumaticaand giant-cell (Takayasu's) arteritis), medium-vessel vasculitis(including Kawasaki's disease and polyarteritis nodosa/periarteritisnodosa), microscopic polyarteritis, immunovasculitis, CNS vasculitis,cutaneous vasculitis, hypersensitivity vasculitis, necrotizingvasculitis such as systemic necrotizing vasculitis, and ANCA-associatedvasculitis, such as Churg-Strauss vasculitis or syndrome (CSS) andANCA-associated small-vessel vasculitis, temporal arteritis, autoimmuneaplastic anemia, Coombs positive anemia, Diamond Blackfan anemia,hemolytic anemia or immune hemolytic anemia including autoimmunehemolytic anemia (AIHA), pernicious anemia (anemia perniciosa),Addison's disease, pure red cell anemia or aplasia (PRCA), Factor VIIIdeficiency, hemophilia A, autoimmune neutropenia, pancytopenia,leukopenia, diseases involving leukocyte diapedesis, CNS inflammatorydisorders, multiple organ injury syndrome such as those secondary tosepticemia, trauma or hemorrhage, antigen-antibody complex-mediateddiseases, anti-glomerular basement membrane disease, anti-phospholipidantibody syndrome, allergic neuritis, Behcet's disease/syndrome,Castleman's syndrome, Goodpasture's syndrome, Reynaud's syndrome,Sjogren's syndrome, Stevens-Johnson syndrome, pemphigoid such aspemphigoid bullous and skin pemphigoid, pemphigus (including pemphigusvulgaris, pemphigus foliaceus, pemphigus mucus-membrane pemphigoid, andpemphigus erythematosus), autoimmune polyendocrinopathies, Reiter'sdisease or syndrome, an immune complex disorder such as immune complexnephritis, antibody-mediated nephritis, polyneuropathies, chronicneuropathy such as IgM polyneuropathies or IgM-mediated neuropathy, andautoimmune or immune-mediated thrombocytopenia such as idiopathicthrombocytopenic purpura (ITP) including chronic or acute ITP, scleritissuch as idiopathic cerato-scleritis, episcleritis, autoimmune disease ofthe testis and ovary including autoimmune orchitis and oophoritis,primary hypothyroidism, hypoparathyroidism, autoimmune endocrinediseases including thyroiditis such as autoimmune thyroiditis,Hashimoto's disease, chronic thyroiditis (Hashimoto's thyroiditis), orsubacute thyroiditis, idiopathic hypothyroidism, Grave's disease,polyglandular syndromes such as autoimmune polyglandular syndromes (orpolyglandular endocrinopathy syndromes), paraneoplastic syndromes,including neurologic paraneoplastic syndromes such as Lambert-Eatonmyasthenic syndrome or Eaton-Lambert syndrome, stiff-man or stiff-personsyndrome, encephalomyelitis such as allergic encephalomyelitis orencephalomyelitis allergica and experimental allergic encephalomyelitis(EAE), myasthenia gravis such as thymoma-associated myasthenia gravis,cerebellar degeneration, neuromyotonia, opsoclonus or opsoclonusmyoclonus syndrome (OMS), and sensory neuropathy, multifocal motorneuropathy, Sheehan's syndrome, autoimmune hepatitis, lupoid hepatitis,giant-cell hepatitis, autoimmune chronic active hepatitis, lymphoidinterstitial pneumonitis (LIP), bronchiolitis obliterans(non-transplant) vs NSIP, Guillain-Barre syndrome, Berger's disease (IgAnephropathy), idiopathic IgA nephropathy, linear IgA dermatosis, acutefebrile neutrophilic dermatosis, subcorneal pustular dermatosis,transient acantholytic dermatosis, cirrhosis such as primary biliarycirrhosis and pneumonocirrhosis, autoimmune enteropathy syndrome, Celiacor Coeliac disease, celiac sprue (gluten enteropathy), refractory sprue,idiopathic sprue, cryoglobulinemia, amylotrophic lateral sclerosis (ALS;Lou Gehrig's disease), coronary artery disease, autoimmune ear diseasesuch as autoimmune inner ear disease (AIED), autoimmune hearing loss,polychondritis such as refractory or relapsed or relapsingpolychondritis, pulmonary alveolar proteinosis, Cogan'ssyndrome/nonsyphilitic interstitial keratitis, Bell's palsy, Sweet'sdisease/syndrome, rosacea autoimmune, zoster-associated pain,amyloidosis, a non-cancerous lymphocytosis, a primary lymphocytosis,which includes monoclonal B cell lymphocytosis (e.g., benign monoclonalgammopathy and monoclonal gammopathy of undetermined significance,MGUS), peripheral neuropathy, paraneoplastic syndrome, channelopathiesincluding channelopathies of the CNS, autism, inflammatory myopathy,focal or segmental or focal segmental glomerulosclerosis (FSGS),endocrine opthalmopathy, uveoretinitis, chorioretinitis, autoimmunehepatological disorder, fibromyalgia, multiple endocrine failure,Schmidt's syndrome, adrenalitis, gastric atrophy, presenile dementia,demyelinating diseases such as autoimmune demyelinating diseases andchronic inflammatory demyelinating polyneuropathy, Dressler's syndrome,alopecia areata, alopecia totalis, CREST syndrome (calcinosis, Raynaud'sphenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia),male and female autoimmune infertility, e.g., due to anti-spermatozoanantibodies, mixed connective tissue disease, Chagas' disease, rheumaticfever, recurrent abortion, farmer's lung, erythema multiforme,post-cardiotomy syndrome, Cushing's syndrome, bird-fancier's lung,allergic granulomatous angiitis, benign lymphocytic angiitis, Alport'ssyndrome, alveolitis such as allergic alveolitis and fibrosingalveolitis, interstitial lung disease, transfusion reaction, Sampter'ssyndrome, Caplan's syndrome, endocarditis, endomyocardial fibrosis,diffuse interstitial pulmonary fibrosis, interstitial lung fibrosis,pulmonary fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis,endophthalmitis, erythema elevatum et diutinum, erythroblastosisfetalis, eosinophilic faciitis, Shulman's syndrome, Felty's syndrome,cyclitis such as chronic cyclitis, heterochronic cyclitis, iridocyclitis(acute or chronic), or Fuch's cyclitis, Henoch-Schonlein purpura, SCID,sepsis, endotoxemia, post-vaccination syndromes, Evan's syndrome,autoimmune gonadal failure, Sydenham's chorea, post-streptococcalnephritis, thromboangitis ubiterans, thyrotoxicosis, tabes dorsalis,chorioiditis, giant-cell polymyalgia, chronic hypersensitivitypneumonitis, keratoconjunctivitis sicca, idiopathic nephritic syndrome,minimal change nephropathy, benign familial and ischemia-reperfusioninjury, transplant organ reperfusion, retinal autoimmunity, aphthae,aphthous stomatitis, arteriosclerotic disorders, aspermiogenesis,autoimmune hemolysis, Boeck's disease, enteritis allergica, erythemanodosum leprosum, idiopathic facial paralysis, chronic fatigue syndrome,febris rheumatica, Hamman-Rich's disease, sensoneural hearing loss,ileitis regionalis, leucopenia, transverse myelitis, primary idiopathicmyxedema, ophthalmia symphatica, polyradiculitis acuta, pyodermagangrenosum, acquired spenic atrophy, vitiligo, toxic-shock syndrome,conditions involving infiltration of T cells, leukocyte-adhesiondeficiency, immune responses associated with acute and delayedhypersensitivity mediated by cytokines and T-lymphocytes, diseasesinvolving leukocyte diapedesis, multiple organ injury syndrome,antigen-antibody complex-mediated diseases, antiglomerular basementmembrane disease, allergic neuritis, autoimmune polyendocrinopathies,oophoritis, primary myxedema, autoimmune atrophic gastritis, rheumaticdiseases, mixed connective tissue disease, nephrotic syndrome,insulitis, polyendocrine failure, autoimmune polyglandular syndrome typeI, adult-onset idiopathic hypoparathyroidism (AOIH), myocarditis,nephrotic syndrome, primary sclerosing cholangitis, acute or chronicsinusitis, ethmoid, frontal, maxillary, or sphenoid sinusitis, aneosinophil-related disorder such as eosinophilia, pulmonary infiltrationeosinophilia, eosinophilia-myalgia syndrome, Loffler's syndrome, chroniceosinophilic pneumonia, tropical pulmonary eosinophilia, granulomascontaining eosinophils, seronegative spondyloarthritides, polyendocrineautoimmune disease, sclerosing cholangitis, sclera, episclera, Bruton'ssyndrome, transient hypogammaglobulinemia of infancy, Wiskott-Aldrichsyndrome, ataxia telangiectasia syndrome, angiectasis, autoimmunedisorders associated with collagen disease, rheumatism, allergichypersensitivity disorders, glomerulonephritides, reperfusion injury,ischemic reperfusion disorder, lymphomatous tracheobronchitis,inflammatory dermatoses, dermatoses with acute inflammatory components,and autoimmune uveoretinitis (AUR).

In another embodiment of the above described methods, the method furthercomprises identifying a subject who is at risk of developing T1D, so asto prevent or delay onset of diabetes symptoms. For example, anindividual who has detectable amount of self-autoantibodies associatedwith T1D that is known in the art. See the risk factors and markersdescribed by Ping Xu and Jeffrey P. Krischer in “Prognosticclassification factors associated with development of multipleautoantibodies, dysglycemia, and Type 1 Diabetes—A recursivepartitioning analysis” in Diabetes Care, 2016, 39(6): 1036-1044.

In one embodiment of any one of the method described, the autoimmunedisorder is Type 1 diabetes (T1D).

In one embodiment of any one of the method described, the subject hasbeen newly diagnosed with T1D.

In one embodiment of any one of the method described, the subject hasbeen newly been detected to have self-autoantibodies associated withT1D, e.g., GAD65 autoantibody, and islet antigen 2 autoantibody.

In one embodiment of any one of the method described, the HSCs areautologous to the recipient subject.

In one embodiment of any one of the method described, the HSCs arenon-autologous and allogenic to the recipient subject.

In one embodiment of any one of the method described, the HSCs arenon-autologous and xenogeneic to the recipient subject.

In one embodiment of any one of the method described, the population ofHSCs is obtained from the bone marrow, umbilical cord, amniotic fluid,chorionic villi, cord blood, placental blood or peripheral blood.

In one embodiment of any one of the method described, the population ofHSCs is obtained from mobilized peripheral blood.

In one embodiment of any one of the method described, the population ofHSCs comprises CD34⁺ cells. In another embodiment, the population ofHSCs comprises CD34⁺ selected cells obtained from the bone marrow,umbilical cord, amniotic fluid, chorionic villi, cord blood, placentalblood or peripheral blood or mobilized peripheral blood.

In one embodiment of any one of the method described, the population ofHSCs is autologous to the recipient subject.

In one embodiment of any one of the method described, the population ofHSCs is at the minimum HLA type matched to the recipient subject.

In one embodiment of any one of the method described, the population ofHSCs are cryopreserved after the removal of excess PGE₂ or afterpost-transfection with the vector, ex vivo cultured to expand thepopulation of modified HSCs, prior to transplantation into the recipientsubject.

In other embodiments of the compositions and methods described herein,the HSCs can be ex vivo culture expanded any time to increase the numberof starting HSCs for transduction with a vector described herein orstimulation with PGE₂ or for use in therapy. For example, ex vivoculture cell expansion can take place after harvesting from a donorsubject, after transduction with the vector described herein, aftercontact with PGE₂, after any cryopreservation step described herein.

In other embodiments of the compositions and methods described herein,cryopreservation of the HSCs can take place any time after harvestingfrom a donor subject, after culture expansion following harvesting froma donor subject, after transduction with the vector described herein,after contact with PGE₂, after the removal of excess PGE₂, after cultureexpansion following transduction with the vector described herein orafter contact with PGE₂.

In one embodiment of any one of the method described, the population ofHSCs are ex vivo culture expanded after the removal of excess PGE₂ orafter post-transfection with the vector, prior to transplantation intothe recipient subject.

In one embodiment of any one of method described herein, after thecontacting, the HSC is cryopreserved prior to use, for example, ex vivoexpansion and/or implantation into a subject.

In one embodiment of any one of the method described herein, after thecontacting, the HSC is culture expanded ex vivo prior to use, forexample, cryopreservation, and/or implantation/engraftment into asubject.

In one embodiment of any one of the method described, the method furthercomprises identifying a subject afflicted with an autoimmune disease ordisorder or an individual who is an organ or bone marrow transplantrecipient.

In another embodiment of any one of the method described, the methodfurther comprises selecting a subject having an autoimmune disease ordisorder or an individual who is an organ or bone marrow transplantrecipient.

In one embodiment of any one of the method described, the method furthercomprises selecting a recipient subject in need of immune responsemodulation. Such as an individual who is an organ or bone marrowtransplant recipient who has received an allogenic graft.

In another embodiment of any one of the method described, the methodfurther comprises identifying a subject in need of immune responsesuppression. Such as an individual who is an organ or bone marrowtransplant recipient who has received an allogenic graft.

In another embodiment of any one of the method described, the methodfurther comprises selecting a subject in need of immune responsesuppression. Such as an individual who is an organ or bone marrowtransplant recipient who has received an allogenic graft.

In one embodiment of any one of the method described, the method furthercomprises allowing the population of PD-L1⁺ expressing HSCs todifferentiate in vivo into PD-L1⁺ expressing progeny cells.

In one embodiment of any one of the method described herein, after thecontacting, the HSC is differentiated in culture ex vivo prior to use,for example, cryopreservation, and/or implantation/engraftment into asubject.

In one embodiment of any one of the therapeutic method described herein,the chemotherapy and/or radiation is to reduce endogenous stem cells tofacilitate engraftment and/or reconstitution of the implanted cells.

In one aspect of any one of the method described herein, the PD-L1expressing HSCs or progeny cells thereof are further treated ex vivowith prostaglandin E2 and/or antioxidant N-acetyl-L-cysteine (NAC) topromote subsequent engraftment and/or reconstitution of the cells whenimplanted in a recipient subject.

In one embodiment of any one of the method described, the method furthercomprises administering an additional immunosuppression therapy to thesubject.

In one embodiment of any one of the method described, the additionalimmunosuppression therapy comprises thymoglobulin, cyclophosphamide, orboth thymoglobulin plus cyclophosphamide.

In one embodiment of any one of the method described, the additionalimmunosuppression therapy comprises antithymocyte antigens (ATG), orCTLA4-fusion immunoglobulins, or both.

In some embodiments, non-limiting examples of additionalimmunosuppression therapy are calcineurin inhibitors (such ascyclosporine, voclosporin and tacrolimus); CD80/86:CD28 costimulationinhibitors (CTLA4-fusion immunoglobulins such as abatacept andbelatacept); CD154:CD40 costimulation inhibitors (anti-CD40 monoclonalantibodies such as ASKP1240; Astellas); CD20 inhibitors (anti-CD20antibodies such as rituximab, ocrelizumab, ofatumumab, and veltuzumab);CD22 inhibitors (anti-CD22 antibodies such as epratuzumab); B celldifferentiation inhibitors (such as belimumab and atacicept);antibody-producing plasma cell inhibitors (such as bortezomib);inhibitor of the complement process (such as eculizumab); inhibitors ofcytokines that are involved in the immune response with the T or B cells(such as steroids e.g. dexamethasome, glucocorticoid and corticosteroid;Janus kinase inhibitor e.g. tofacitinib; IL-6 receptor inhibitor, e.g.basiliximab; TNF inhibitors e.g. infliximab, adalimumab, golimumab, andcertolizumab; IL-1 inhibitors e g anakinra, rilonacept, and canakinumab;and IL-17 inhibitor e.g. secukinumab); inhibitors of chemokines and celladhesion (such as CCR5 receptor antagonist maraviroc, CXCR4 antagonistplerixafor, CCR4 humanized mAb mogamulizumab, and CCL2 (also known asmonocyte chemotactic protein 1) inhibitor emapticap; pooled intravenousimmunoglobulins (IVIG) from several thousand plasma donors; polyclonalantithymocyte globulin (ALG) and antithymocyte antigens (ATG); CD52inhibitors (anti-CD25 e.g. alemtuzumab); mTOR inhibitors (e.g.,rapamycin, sirolimus and everolimus); and other anti-metabolites such asDNA synthesis inhibitor e.g., azathioprine (AZA), mycophenolate,leflunomide, and cytotoxic agents such as cyclophosphamide.

Lentiviral vectors of the disclosure include, but are not limited to,human immunodeficiency virus (e.g., HIV-1, HIV-2), felineimmunodeficiency virus (FIV), simian immunodeficiency virus (SIV),bovine immunodeficiency virus (BIV), and equine infectious anemia virus(EIAV). These vectors can be constructed and engineered usingart-recognized techniques to increase their safety for use in therapyand to include suitable expression elements and therapeutic genes, suchas described above.

In consideration of the potential toxicity of virus-based vectors, thevectors can be designed in different ways to increase their safety ingene therapy applications. For example, the vector can be made safer byseparating the necessary lentiviral genes (e.g., gag and pol) ontoseparate vectors as described, for example, in U.S. Pat. No. 6,365,150,the contents of which are incorporated by reference herein. Thus,recombinant retrovirus can be constructed such that the retroviralcoding sequence (gag, pol, env) is replaced by a gene of interestrendering the retrovirus replication defective. The replicationdefective retrovirus is then packaged into virions through the use of ahelper virus or a packaging cell line, by standard techniques. Protocolsfor producing recombinant retroviruses and for infecting cells in vitroor in vivo with such viruses can be found in Current Protocols inMolecular Biology, Ausubel, F. M. et al. (eds.) Greene PublishingAssociates, (1989), Sections 9.10-9.14 and other standard laboratorymanuals.

A major prerequisite for the use of viruses as gene delivery vectors isto ensure the safety of their use, particularly with regard to thepossibility of the spread of wild-type virus in the cell population. Thedevelopment packaging cell lines, which produce onlyreplication-defective retroviruses, has increased the utility ofretroviruses for gene therapy, and defective retroviruses are wellcharacterized for use in gene transfer for gene therapy purposes (for areview see Miller, A. D. (1990) Blood 76:271). Accordingly, in oneembodiment of the disclosure, packaging cell lines are used to propagatevectors (e.g., lentiviral vectors) of the disclosure to increase thetiter of the vector virus. The use of packaging cell lines is alsoconsidered a safe way to propagate the virus, as use of the systemreduces the likelihood that recombination will occur to generatewild-type virus. In addition, to reduce toxicity to cells that caused byexpression of packaging proteins, packaging systems can be use in whichthe plasmids encoding the packaging functions of the virus are onlytransiently transfected by, for example, chemical means.

In another embodiment, the vector can be made safer by replacing certainlentiviral sequences with non-lentiviral sequences. Thus, lentiviralvectors of the present disclosure may contain partial (e.g., split) genelentiviral sequences and/or non-lentiviral sequences (e.g., sequencesfrom other retroviruses) as long as its function (e.g., viral titer,infectivity, integration and ability to confer high levels and durationof therapeutic gene expression) are not substantially reduced. Elementswhich may be cloned into the viral vector include, but are not limitedto, promoter, packaging signal, LTR(s), polypurine tracts, and a reverseresponse element (RRE).

In one embodiment of the disclosure, the LTR region is modified byreplacing the viral LTR promoter with a heterologous promoter. In oneembodiment, the promoter of the 5′ LTR is replaced with a heterologouspromoter. Examples of heterologous promoters which can be used include,but are not limited to, a spleen focus-forming virus (SFFV) promoter, atetracycline-inducible (TET) promoter, a β-globin locus control regionand a β-globin promoter (LCR), and a cytomegalovirus (CMV) promoter. Insome embodiments, the promoter is a regulatable promoter, an induciblepromoter, for the regulating the production of PD-L1. For example, aTetracyclin-inducible or Doxycyclin-inducible promoter.

In some embodiments, the viral vectors such as lentiviral vector or AAVor avian viral vectors of the disclosure also include vectors which havebeen modified to improve upon safety in the use of the vectors as genedelivery agents in gene therapy. In one embodiment of this disclosure,an LTR region, such as the 3′ LTR, of the vector is modified in the U3and/or U5 regions, wherein a SIN vector is created. Such modificationscontribute to an increase in the safety of the vector for gene deliverypurposes. In one embodiment, the vector comprises a deletion in the 3′LTR wherein a portion of the U3 region is replaced with an insulatorelement. The insulator prevents the enhancer/promoter sequences withinthe vector from influencing the expression of genes in the nearbygenome, and vice/versa, to prevent the nearby genomic sequences frominfluencing the expression of the genes within the vector. In a furtherembodiment of this disclosure, the 3′ LTR is modified such that the U5region is replaced, for example, with an ideal poly(A) sequence. Itshould be noted that modifications to the LTRs such as modifications tothe 3′ LTR, the 5′ LTR, or both 3′ and 5′ LTRs, are also included in thedisclosure.

The promoter of the lentiviral vector can be one which is naturally(i.e., as it occurs with a cell in vivo) or non-naturally associatedwith the 5′ flanking region of a particular gene. Promoters can bederived from eukaryotic genomes, viral genomes, or synthetic sequences.Promoters can be selected to be non-specific (active in all tissues)(e.g., SFFV), tissue specific (e.g., (LCR), regulated by naturalregulatory processes, regulated by exogenously applied drugs (e.g.,TET), or regulated by specific physiological states such as thosepromoters which are activated during an acute phase response or thosewhich are activated only in replicating cells. Non-limiting examples ofpromoters in the present disclosure include the spleen focus-formingvirus promoter, a tetracycline-inducible promoter, a β-globin locuscontrol region and a β-globin promoter (LCR), a cytomegalovirus (CMV)promoter, retroviral LTR promoter, cytomegalovirus immediate earlypromoter, SV40 promoter, and dihydrofolate reductase promoter. Thepromoter can also be selected from those shown to specifically expressin the select cell types such as HSCs and their progenies. In oneembodiment, the promoter of the vector is cell specific such that geneexpression is restricted to red blood cells. Erythrocyte-specificexpression is achieved by using the human β-globin promoter region andlocus control region (LCR).

Skilled practitioners will recognize that selection of the promoter toexpress the polynucleotide of interest will depend on the vector, thenucleic acid cassette, the cell type to be targeted, and the desiredbiological effect. Skilled practitioners will also recognize that in theselection of a promoter, the parameters can include: achievingsufficiently high levels of gene expression to achieve a physiologicaleffect; maintaining a critical level of gene expression; achievingtemporal regulation of gene expression; achieving cell type specificexpression; achieving pharmacological, endocrine, paracrine, orautocrine regulation of gene expression; and preventing inappropriate orundesirable levels of expression. Any given set of selectionrequirements will depend on the conditions but can be readily determinedonce the specific requirements are determined. In one embodiment of thisdisclosure, the promoter is cell-specific such that gene expression isrestricted to red blood cells. Erythrocyte-specific expression isachieved by using the human β-globin promoter region and locus controlregion (LCR).

Standard techniques for the construction of expression vectors suitablefor use in the present disclosure are well-known to those of ordinaryskill in the art and can be found in such publications as Michael R.Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012). Avariety of strategies are available for ligating fragments of DNA, thechoice of which depends on the nature of the termini of the DNAfragments and which choices can be readily made by the skilled artisan.

The step of facilitating the production of infectious viral particles inthe cells may be carried out using conventional techniques, such asstandard cell culture growth techniques. If desired by the skilledpractitioner, lentiviral stock solutions may be prepared using thevectors and methods of the present disclosure. Methods of preparingviral stock solutions are known in the art and are illustrated by, e.g.,Y. Soneoka et al. (1995) Nucl. Acids Res. 23:628-633, and N. R. Landauet al. (1992) J. Virol. 66:5110-5113. In the method of producing a stocksolution in the present disclosure, lentiviral-permissive cells(referred to herein as producer cells) are transfected with the vectorsystem of the present disclosure. The cells are then grown undersuitable cell culture conditions, and the lentiviral particles collectedfrom either the cells themselves or from the cell media as describedabove. Suitable producer cell lines include, but are not limited to, thehuman embryonic kidney cell line 293, the equine dermis cell line NBL-6,and the canine fetal thymus cell line Cf2TH.

The step of collecting the infectious virus particles also can becarried out using conventional techniques. For example, the infectiousparticles can be collected by cell lysis, or collection of thesupernatant of the cell culture, as is known in the art. Optionally, thecollected virus particles may be purified if desired. Suitablepurification techniques are well known to those skilled in the art.

Other methods relating to the use of viral vectors in gene therapy canbe found in, e.g., Kay, M. A. (1997) Chest 111(6 Supp.):1385-1425;Ferry, N. and Heard, J. M. (1998) Hum. Gene Ther. 9:1975-81; Shiratory,Y. et al. (1999) Liver 19:265-74; Oka, K. et al. (2000) Curr. Opin.Lipidol. 11:179-86; Thule, P. M. and Liu, J. M. (2000) Gene Ther.7:1744-52; Yang, N. S. (1992) Crit. Rev. Biotechnol. 12:335-56; Alt, M.(1995) J. Hepatol. 23:746-58; Brody, S. L. and Crystal, R. G. (1994)Ann. N.Y. Acad. Sci. 716:90-101; Strayer, D. S. (1999) Expert Opin.Investig. Drugs 8:2159-2172; Smith-Arica, J. R. and Bartlett, J. S.(2001) Curr. Cardiol. Rep. 3:43-49; and Lee, H. C. et al. (2000) Nature408:483-8.

In one embodiment, the sample of HSCs is contacted with at least 10³vectors or viral vectors or particles per 10⁶ HSC cells in the ex vivotransfection or transduction procedure. The vector carries an exogenouscopy of a nucleic acid encoding a PD-L1. Other vector dosage ranges setforth herein for contacting with the sample of HSCs is exemplary onlyand are not intended to limit the scope or practice of the claimedcomposition or methods described herein. In one embodiment, the vectordosage is ranges from 10³-10⁸ viral particles/10⁶ HSC cells. In otherembodiments, the vector dosage is ranges from 10³-10⁵ viralparticles/10⁶ HSC cells, 10⁴-10⁶ viral particles/10⁶ HSC cells, 10⁵-10⁷viral particles/10⁶ HSC cells, 10³-10⁸ viral particles/10⁶ HSC cells. Inone embodiment, the dosage is about 10⁴ viral particles/10⁶ HSC cells.

The retroviral or lentiviral vectors, or avian viral vector oradeno-associated viral vectors are ex vivo contacted with the HSCs usingstandard transfection techniques well known in the art.

In one embodiment, the retroviral or lentiviral vectors or avian viralvector or adeno-associated viral vectors are transduced into HSCs,hematopoietic progenitor cells or precursors of erythrocytes.

Another aspect of the disclosure pertains to compositions comprising thevectors described. In one embodiment, the composition includes alentiviral vector or avian viral vector or adeno-associated viralvectors in an effective amount sufficient to transduce a sample of HSCsand a pharmaceutically acceptable carrier. An “effective amount” withrespect to vector transduction refers to an amount effective, at dosagesand for periods of time necessary, to achieve the desired result ofintroducing the exogenous PD-L1 encoding nucleic acid into HSCs. Aneffective amount of viral vector may vary according to factors such asthe disease state, age, sex, and weight of the donor individual, and theability of the viral vector to elicit a desired response in thetransduced HSCs. Dosage regimens may be adjusted to provide the optimumresponse. An effective amount is also one in which any toxic ordetrimental effects of the viral vector are outweighed by the beneficialeffects. The potential toxicity of the viral vectors of the disclosurecan be assayed using cell-based assays or art recognized animal modelsand an effective modulator can be selected which does not exhibitsignificant toxicity.

Sterile solutions can be prepared by incorporating lentiviral vector inthe required amount in an appropriate solvent with one or a combinationof ingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating theactive compound into a sterile vehicle which contains a basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile solutions,the preferred methods of preparation are vacuum drying and freeze-dryingwhich yields a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.

In some embodiments, the PD-L1⁺ HSC cells or compositions comprising thePD-L1⁺ HSC cells are sterile and are formulated for therapy in asubject. In one embodiment, the subject is a mammal, e.g., a human.

In some embodiments, the PD-L1⁺ HSC cells or compositions comprising thePD-L1⁺ HSC cells comprise serum or plasma. Alternatively, thecompositions comprise a cryopreservative, e.g., DMSO.

In some embodiments, the compositions or pharmaceutical compositions areformulated for systemic delivery. In some embodiments, the compositionscan be formulated for delivery to specific organs, for example but notlimited to the liver, spleen, the bone marrow, and the skin.Pharmaceutical compositions comprise pharmaceutically acceptablecarrier.

In addition, the compositions or pharmaceutical compositions describedherein can be administered together with other components ofbiologically active agents, such as pharmaceutically acceptablesurfactants (e.g., glycerides), excipients (e.g., lactose), carriers,serum, plasma, diluents and vehicles.

In some embodiments, the compositions or pharmaceutical compositionsdescribed herein contain about 1×10⁶ cells to about 3×10⁶ cells; about1.0×10⁶ cells to about 5×10⁶ cells; about 1.0×10⁶ cells to about 10×10⁶cells, about 10×10⁶ cells to about 20×10⁶ cells, about 10×10⁶ cells toabout 30×10⁶ cells, or about 20×10⁶ cells to about 30×10⁶ PD-L1expressing cells or HSCs or their progeny.

In some embodiments, the compositions or pharmaceutical compositionsdescribed herein contain about 1×10⁶ cells to about 30×10⁶ cells; about1.0×10⁶ cells to about 20×10⁶ cells; about 1.0×10⁶ cells to about 10×10⁶cells, about 2.0×10⁶ cells to about 30×10⁶ cells, about 2.0×10⁶ cells toabout 20×10⁶ cells, or about 2.0×10⁶ cells to about 10×10⁶ PD-L1expressing cells or HSCs or their progeny.

In some embodiments, the compositions or pharmaceutical compositionsdescribed herein contain about 1×10⁶ hematopoietic stem or progenitorcells, about 2×10⁶ cells, about 5×10⁶ cells, about 7×10⁶ cells, about10×10⁶ cells, about 15×10⁶ cells, about 17×10⁶ cells, about 20×10⁶ cellsabout 25×10⁶ cells, or about 30×10⁶ PD-L1 expressing cells or HSCs ortheir progeny.

The dosage of PD-L1⁺ HSC cells administered to a recipient subject willvary depending upon a variety of factors, including the number of PD-L1⁺HSCs available, the level of expression of PD-L1 in the HSCs, route ofadministration, size, age, sex, health, body weight and diet of therecipient; nature and extent of symptoms of the disease being treated,kind of concurrent treatment, frequency of treatment, and the effectdesired.

In one embodiment, the dosage of PD-L1 expressing HSCs should be largeenough a cell population transplanted to ensure sufficient engraftmentand reconstitution in vivo after implantation into the subject.

In one embodiment, the dosage is at least 1×10⁴ cells per implantation.In other embodiments, the dosage is at least 5×10⁴ cells, at least 1×10⁵cells, at least 5×10⁵ cells, at least 1×10⁶ cells, at least 5×10⁶ cells,at least 1×10⁷ cells, at least 5×10⁷ cells, at least 1×10⁸ cells, atleast 5×10⁸ cells, at least 1×10⁹ cells, at least 5×10⁹ cells, or atleast 1×10¹⁰ cells or more per implantation into a subject. Second orsubsequent administrations can be administered at a dosage which is thesame, less than or greater than the initial or previous doseadministered to the individual.

In some embodiments, the dosage of PD-L1⁺ HSC cells administered to arecipient subject is about at least 0.1×10⁵ cells/kg of bodyweight, atleast 0.5×10⁵ cells/kg of bodyweight, at least 1×10⁵ cells/kg ofbodyweight, at least 5×10⁵ cells/kg of bodyweight, at least 10×10⁵cells/kg of bodyweight, at least 0.5×10⁶ cells/kg of bodyweight, atleast 0.75×10⁶ cells/kg of bodyweight, at least 1×10⁶ cells/kg ofbodyweight, at least 1.25×10⁶ cells/kg of bodyweight, at least 1.5×10⁶cells/kg of bodyweight, at least 1.75×10⁶ cells/kg of bodyweight, atleast 2×10⁶ cells/kg of bodyweight, at least 2.5×10⁶ cells/kg ofbodyweight, at least 3×10⁶ cells/kg of bodyweight, at least 4×10⁶cells/kg of bodyweight, at least 5×10⁶ cells/kg of bodyweight, at least10×10⁶ cells/kg of bodyweight, at least 15×10⁶ cells/kg of bodyweight,at least 20×10⁶ cells/kg of bodyweight, at least 25×10⁶ cells/kg ofbodyweight, or at least 30×10⁶ cells/kg of bodyweight of the subjectrecipient.

In another embodiment, the dosage is at least 2×10⁶ cells/kg bodyweightof the recipient subject. In other embodiments, the dosage is at least3×10⁶ cells/kg of bodyweight, at least 4×10⁶ cells/kg of bodyweight, atleast 5×10⁶ cells/kg of bodyweight, at least 6×10⁶ cells/kg ofbodyweight, at least 7×10⁶ cells/kg of bodyweight, at least 8×10⁶cells/kg of bodyweight, at least 9×10⁶ cells/kg of bodyweight, at least10×10⁶ cells/kg of bodyweight, at least 15×10⁶ cells/kg of bodyweight,at least 20×10⁶ cells/kg of bodyweight, at least 25×10⁶ cells/kg ofbodyweight, or at least 30×10⁶ cells/kg of bodyweight of the subjectrecipient.

In another embodiment, the dosage is at least greater than 5×10⁶cells/kg bodyweight of the recipient subject.

In another embodiment, the dosage is at least greater than 10×10⁶cells/kg bodyweight of the recipient subject.

A second or subsequent administration is preferred. For example, secondand subsequent administrations can be given between about one day to 30weeks from the previous administration. Two, three, four or more totaladministrations can be delivered to the individual, as needed.

The precise dose to be employed in the formulation will also depend onthe route of administration, and the seriousness of the disease ordisorder, and should be decided according to the judgment of thepractitioner and each patient's circumstances. Effective doses may beextrapolated from dose-response curves derived from in vitro or animalmodel test systems.

Efficacy testing can be performed during the course of treatment usingthe methods described herein. Measurements of the degree of severity ofa number of symptoms associated with a particular ailment are notedprior to the start of a treatment and then at later specific time periodafter the start of the treatment. For example, the amount of insulin inthe blood or blood glucose after a meal.

The present disclosure can be defined in any of the following numberedparagraphs:

-   -   [1] A population of modified HSCs where the cells carry an        exogenous copy of a nucleic acid encoding a PD-L1.    -   [2] The population of modified HSCs of paragraph 1, wherein the        cells are expressing PD-L1.    -   [3] The population of modified HSCs of paragraph 1 or 2, wherein        the nucleic acid is a cDNA.    -   [4] The population of modified HSCs of paragraph 1 or 2, wherein        the nucleic acid is a genomic DNA.    -   [5] The population of modified HSCs of paragraph 4, wherein the        nucleic acid is integrated into the genome of the cells.    -   [6] The population of modified HSCs of any one of the preceding        paragraphs, wherein the nucleic acid is introduced into the        cells via a vector.    -   [7] The population of modified HSCs of paragraph 6, wherein the        vector is a viral vector.    -   [8] The population of modified HSCs of paragraph 7, wherein the        viral vector is a lentiviral vector.    -   [9] The population of modified HSCs of any one of the preceding        paragraphs, wherein the cells are mammalian cells.    -   [10] The population of modified HSCs of paragraph 9, wherein the        mammalian cells are human cells.    -   [11] The population of modified HSCs of any one of the preceding        paragraphs, wherein prior to the modification, the HSCs are        obtained from the bone marrow, umbilical cord, amniotic fluid,        chorionic villi, cord blood, placental blood or peripheral        blood.    -   [12] The population of modified HSCs of paragraph 11, wherein        the HSCs are obtained from mobilized peripheral blood.    -   [13] The population of modified HSCs of any one of the preceding        paragraphs, wherein the HSCs are derived from a healthy        individual.    -   [14] The population of modified HSCs of any one of the preceding        paragraphs, wherein the HSCs are derived from an individual with        a diagnosed disease or disorder.    -   [15] The population of modified HSCs of paragraph 14, wherein        the diagnosed disease or disorder is an autoimmune disease or        disorder.    -   [16] The population of modified HSCs of paragraph 15, wherein        the autoimmune disease or disorder is Type 1 diabetes (T1D).    -   [17] The population of modified HSCs of any one of the preceding        paragraphs, wherein the cells are ex vivo cultured before or        after or both before and after the introduction of the exogenous        copy of a nucleic acid encoding a PD-L1.    -   [18] The population of modified HSCs of any one of the preceding        paragraphs, wherein the cells are cryopreserved prior to or        after or both before and after the introduction of the exogenous        copy of a nucleic acid encoding a PD-L1.    -   [19] The population of modified HSCs of any one of the preceding        paragraphs, wherein the cells are cryopreserved prior to use.    -   [20] The population of modified HSCs of any one of the preceding        paragraphs, wherein the cells are produced by a method        comprising: (a) contacting a sample of HSCs with a vector        carrying an exogenous copy of a nucleic acid encoding a PD-L1 to        modify the HSCs; ex vivo culturing the resultant modified cells        from the contacting; and (c) establishing the expression of        PD-L1 on the modified HSCs, thereby producing a population of        modified HSCs cells expressing PD-L1.    -   [21] The population of modified HSCs of paragraph 20, wherein        the method further comprises establishing that there is at least        one fold increase in the number of PD-L1+ expressing cells        compared to non-modified cells.    -   [22] An ex vivo method of producing a population of modified,        PD-L1+ expressing HSCs, the method comprising: (a) contacting a        sample of HSCs with a vector carrying an exogenous copy of a        nucleic acid encoding a PD-L1 to modify the HSCs whereby the        exogenous copy of a nucleic acid is introduced into the        HSCs; (b) ex vivo culturing the resultant modified cells from        the contacting; and (c) establishing the expression of PD-L1 on        the modified HSCs, thereby producing a population of modified        HSCs cells expressing PD-L1.    -   [23] The ex vivo method of paragraph 22, wherein the method        further comprises establishing that there is at least one fold        increase in the number of PD-L1+ expressing cells compared to        non-modified cells.    -   [24] The ex vivo method of paragraph 22 or 23, wherein the        sample of HSC is obtained from the bone marrow, umbilical cord,        amniotic fluid, chorionic villi, cord blood, placental blood or        peripheral blood.    -   [25] The ex vivo method of paragraph 24, wherein the sample of        HSC is obtained from mobilized peripheral blood.    -   [26] The ex vivo method of any one of the preceding paragraphs,        wherein the sample of HSCs is obtained from a healthy        individual.    -   [27] The ex vivo method of any one of the preceding paragraphs,        wherein the sample of HSCs is obtained from an individual with a        diagnosed disease or disorder.    -   [28] The ex vivo method of paragraph 27, wherein the diagnosed        disease or disorder is an autoimmune disease or disorder.    -   [29] The ex vivo method of paragraph 28, wherein the autoimmune        disease or disorder is Type 1 diabetes (T1D).    -   [30] The ex vivo method of any one of the preceding paragraphs,        wherein the vector is viral vector.    -   [31] The ex vivo method of paragraph 30, wherein the viral        vector is a lentiviral vector, an avian virus vector or an        adeno-associated virus.    -   [32] The ex vivo method of any one of the preceding paragraphs,        wherein the nucleic acid is a cDNA.    -   [33] The ex vivo method of any one of the preceding paragraphs,        wherein the nucleic acid is a genomic DNA.    -   [34] The ex vivo method of paragraph 33, wherein the nucleic        acid is integrated into the genome of the cells.    -   [35] A composition comprising the hematopoietic stem cells of        any one of the preceding paragraphs or hematopoietic stem cells        produced by any one of the preceding method paragraphs.    -   [36] A composition for transplantation into a subject or for        reducing an immune response in a subject, the composition        comprising the hematopoietic stem cells of any one of the        preceding paragraphs or the hematopoietic stem cells produced by        the method of one of the preceding paragraphs.    -   [37] A method of treating an autoimmune disorder in a subject in        need thereof, the method comprising administering to a subject a        composition comprising the hematopoietic stem cells in any one        of the preceding paragraphs.    -   [38] The method of paragraph 37, wherein the autoimmune disorder        is T1D.    -   [39] The method of paragraph 37 or 38, wherein the HSCs are        autologous to the recipient subject.    -   [40] The method of paragraph 37 or 38, wherein the HSCs are        non-autologous and allogenic to the recipient subject.    -   [41] The method of paragraph 37 or 38, wherein the HSCs are        non-autologous and xenogeneic to the recipient subject.    -   [42] A method of modulating an immune response in a subject        comprising: (a) providing a population of HSCs; (b) contacting        sample of HSCs with prostaglandin E2 (PGE₂) at 0.1 M        concentration for at least 24 hrs at 37° C.; (c) removing the        PGE₂ after 24 hrs, thereby producing a population of PD-L1⁺        expressing HSCs; (d) transplanting said population of PD-L1⁺        expressing HSCs into a recipient subject, thereby modulating the        immune response in the recipient subject.    -   [43] A method of modulating an immune response in a subject        comprising: (a) providing a population of HSCs; (b) contacting        sample of HSCs with a vector carrying an exogenous copy of a        nucleic acid encoding a PD-L1; (c) ex vivo culturing the        resultant modified cells from the contacting; (d) establishing        the expression of PD-L1 on the modified HSCs, thereby producing        a population of modified HSCs cells expressing PD-L1; and (e)        transplanting said population of PD-L1+ expressing HSCs into a        recipient subject, thereby modulating the immune response in the        recipient subject.    -   [44] The method of paragraph 42 or 43, wherein the population of        HSCs is obtained from the bone marrow, umbilical cord, amniotic        fluid, chorionic villi, cord blood, placental blood or        peripheral blood.    -   [45] The method of paragraph 44, wherein the population of HSCs        is obtained from mobilized peripheral blood.    -   [46] The method of any one of the preceding paragraphs, wherein        the population of HSCs autologous to the recipient subject.    -   [47] The method of any one of the preceding paragraphs, wherein        the population of HSCs allogeneic to the recipient subject.    -   [48] The method of any one of the preceding paragraphs, wherein        the population of HSCs is xenogeneic to the recipient subject.    -   [49] The method of any one of the preceding paragraphs, wherein        the population of HSCs are cryopreserved after the removal of        PGE₂ or after ex vivo culturing post-transfection with a vector        prior to transplantation into the recipient subject.    -   [50] The method of claim any one of the preceding paragraphs,        wherein the population of HSCs are culture expanded ex vivo        after the removal of PGE₂ or after ex vivo culturing        post-transfection with a vector prior to transplantation into        the recipient subject.    -   [51] The method of claim any one of the preceding paragraphs,        the method further comprising selecting a recipient subject in        need of immune response modulation.    -   [52] A composition comprising the PD-L1 expressing hematopoietic        stem cells of any one of paragraphs 1-21 or hematopoietic stem        cells produced by any one of the method paragraphs 22-34 for use        in the prevention or treatment of an autoimmune disease or        disorder, for use in suppressing an immune response in a        subject, for use in the delay of the onset of T1D in a subject        at risk of developing T1D, for use in the prevention and delay        of an allogenic tissue or organ transplant rejection, and for        the treatment of T1D in adult and pediatric subjects.    -   [53] A composition comprising the PD-L1 expressing hematopoietic        stem cells of any one of paragraphs 1-21 or hematopoietic stem        cells produced by any one of the method paragraphs 22-34 for the        manufacture of medicament for use in the prevention or treatment        of an autoimmune disease or disorder, in the suppression of an        immune response in a subject, in the delay of the onset of T1D        in a subject at risk of developing T1D, in the prevention and        delay of an allogenic tissue or organ transplant rejection, and        for the treatment of T1D in adult and pediatric subjects.    -   [54] A population of PD-L1 expressing hematopoietic stem cells        of any one of paragraphs 1-21 or hematopoietic stem cells        produced by any one of the method paragraphs 22-34 for use in        the prevention or treatment of an autoimmune disease or        disorder, for use in suppressing an immune response in a        subject, for use in the delay of the onset of T1D in a subject        at risk of developing T1D, for use in the prevention and delay        of an allogenic tissue or organ transplant rejection, and for        the treatment of T1D in adult and pediatric subjects.    -   [55] A population of PD-L1 expressing hematopoietic stem cells        of any one of paragraphs 1-21 or hematopoietic stem cells        produced by any one of the method paragraphs 22-34 for the        manufacture of medicament for use in the prevention or treatment        of an autoimmune disease or disorder, in the suppression of an        immune response in a subject, in the delay of the onset of T1D        in a subject at risk of developing T1D, in the prevention and        delay of an allogenic tissue or organ transplant rejection, and        for the treatment of T1D in adult and pediatric subjects.

The skilled artisan will appreciate that certain factors may influencethe dosage and timing required to effectively treat a subject, includingbut not limited to the severity of the disease or disorder, previoustreatments, the general health and/or age of the subject, and otherdiseases present.

This disclosure is further illustrated by the following example whichshould not be construed as limiting. The contents of all referencescited throughout this application, as well as the figures and table areincorporated herein by reference.

Those skilled in the art will recognize, or be able to ascertain usingnot more than routine experimentation, many equivalents to the specificembodiments of the disclosure described herein. Such equivalents areintended to be encompassed by the following claims.

EXAMPLES Example 1

Exemplary HSCs Ex Vivo Culture Protocol with PGE₂ for Stimulating PD-L1Expression.

CD34⁺ cells were isolated from patients (20 ml of blood) using magneticbeads and ˜1×10⁶ cells were plated in a U-bottom 96-well plate with 200μl of the indicated medium. STFIA medium was defined as serum-freemedium supplemented with 10 μg/ml heparin, 10 ng/ml human SCF, 20 ng/mlhuman TPO, 10 ng/ml human FGF-1, 100 ng/ml IGFBP2, and 500 ng/mlAngptl3. PGE₂ was added the culture at 0 h, 24 h, 72 h and 6 days. Analiquot of 2 μl of diluted PGE₂ at a concentration of 10 μM was added tothe 200 μl of each well in the 96-well plate. The approximate finalconcentration of PGE₂ in each well is 0.1 μM. Therefore, the cells areexposed to −0.1 μM of PGE₂. The periodic addition of PGE₂ at 24 h, 72 hand 6 days serves to maintain the PGE₂ in the culture media. Cells werecultured for 7 days at 37° C. in 5% CO₂ and the normal level of O₂.

Alternatively, cells are cultured in the same conditions for 48 h in theabsence of PGE₂, after which and PGE₂ is added and then later at 24 hafter the initial additional. PGE₂ is added to the same approximatefinal concentration of PGE₂ of 0.1 μM. We typically observed a ˜10-foldincrease of CD34⁺PD-L1⁺ of cells after 8 days of culture when comparedwith a cell culture with the same medium cultured for the same time inthe same conditions with cells obtained from the same subjects butwithout addition of PGE₂.

The percentage of CD34⁺PD-L1⁺ cells obtained at day 0 without culturingin healthy subjects is nearly 24%, in individuals with T1D is 8-10%.This protocol produces increased expression of PD-L1 as compared tobaseline (at least 5-fold increase).

Example 2

In vitro Murine studies—Murine HSCs (Lin⁻c-Kit⁺Sca-1⁺, KLS) expressPD-L1. We evaluated the characteristics of CXCR4 antagonist-mobilizedHSCs by FACS analysis. Lin⁻c-Kit⁺Sca-1⁺ cells were sorted fromislet-transplanted or naïve CXCR4 antagonist-treated mice after 7 and 14days of treatment. Interestingly, while most positive costimulatorymolecules were found to be negative or scarcely expressed (CD40, CD80,CD86, PD-L2, ICOS, OX40, OX40L), PD-L1 was highly expressed by mobilizedHSCs (58.0±7.1%). Extracted HSCs also expressed CXCR4 (38.4±4.2%). Wethen evaluated whether HSC mobilization increases the generation ofPD-L1⁺ HSCs, and PD-L1⁺ HSCs did not increase in bone marrow from B6islet-transplanted mice 6 h after the initiation of CXCR4 antagonisttreatment. PD-L1 genetic deletion abrogates HSC immunoregulatoryproperties. To evaluate the immunoregulatory role of PD-L1 in murineHSCs, we investigated the effect of mobilized HSCs from WT and PD-L1 KOmice on the alloimmune response in vitro. A standard MLR assay wasperformed in which HSCs (from WT B6 or PD-L1 KO mice) were syngeneic toresponder cells (CD4⁺ cells from B6) but allogeneic to bonemarrow-derived DCs (from BALB/c). While HSCs from WT B6 mice abrogatedthe MLR response when added to culture, HSCs from PD-L1 KO mice failedto do so (FIG. 1 ). The percentage of peripheral PD-L1⁺ HSCs is reducedin NOD mice compared to B6. The percentage of peripheral PD-L1⁺ KLS in10-week-old NOD and B6 mice was evaluated by FACS. A reduction of PD-L1⁺KLS was evident in normoglycemic NOD (10-week-old) as compared to B6mice (peripheral PD-L1⁺ HSCs: B6=55.6±1.8 vs. NOD=29.5±1.54%; p<0.001).Moreover, PCR analysis confirmed that PD-L1 mRNA was upregulated in HSCsobtained from B6 mice compared to those obtained from NOD mice (FIGS.2A-2C). The murine PD-L1 defect on HSCs can be overturned in vitro bypharmacologic approach. We performed a pilot study to assess thefeasibility of generation of PD-L1⁺ HSCs in vitro by pharmacologicapproach. KLS cells were isolated from splenocytes of B6 and NOD mice bymagnetic beads and cultured with standard stem cell medium plus PGE₂ (2μl, 10 μM). After 8 days, a ˜30% fold increase in the PD-L1⁺ HSCs wasevident (p=0.002 and p=0.001, in B6 and NOD KLS respectively), (FIGS.2D-2E).

In vitro human cell studies—The percentage of peripheral PD-L1⁺ HSCs isreduced in T1D individuals as compared to healthy subjects. CD34⁺ cellswere successfully purified by magnetic beads and we obtained apercentage of CD34⁺ cells from peripheral blood mononuclear cells(PBMCs) in healthy controls and T1D individuals of 0.05-0.07%. FewerPD-L1⁺ CD34⁺ cells were detectable in T1D individuals as compared tohealthy subjects (T1D=9.5% vs. controls=23.5%; p<0.001), (FIGS. 3A-3C).A PCR analysis performed on RNA extracted from CD34⁺ cells previouslyisolated from PBMCs, confirmed that PD-L1 was upregulated in HSCsobtained from healthy subjects as compared to those obtained from T1Dindividuals. The human PD-L1 defect on HSCs can be overturned in vitroby pharmacologic approach. We performed a pilot study to assess thefeasibility of generation of PD-L1⁺ HSCs ex vivo by a pharmacologicapproach. PBMCs were isolated from peripheral blood of T1D individuals(n=10) using the Ficoll-Plaque protocol and CD34⁺ cells were sorted bymagnetic beads. CD34⁺ cells were cultured as described herein thisdisclosure. After 7 days, a ˜8 times fold increase in the percentage ofPD-L1⁺ HSCs was evident (p=0.001), (FIGS. 3D-3E).

Example 3

In vivo murine studies—PD-L1/PD-1 crosslinking delays diabetes onset inNOD mice and islet graft rejection in streptozotocinated B6 mice. Weused an anti-PD-1 mAb (hybridoma PIM-2, rat IgG2a) recently developed tostimulate PD-1, thus mimicking PD-L1 crosslinking to PD-1. PIM2 Abdelayed the onset of diabetes in NOD mice and islet allograft rejection(BALB/c into B6), (FIGS. 4A-4B). Infusion of PD-L1 transduced KLSreverted hyperglycemia in NOD mice. KLS were isolated from bone marrowof NOD mice and were transduced with PD-L1 pseudoviral particlespreviously obtained by infecting with a lentivirus vector, expressing afluorescent marker ZsGreen and PD-L1 gene, 293TN producer cells. Afterobtaining a high concentration of the virus, KLS can be infected andsubsequently expanded as PD-L1 transduced KLS in a 7-day culture.Expression of PD-L1 was under the control of a doxa promoter, thusdoxacyclin needs to be injected in order to stimulate PD-L1 expressionon transduced cells. 5×10⁶ PD-L1 transduced KLS were then injectedintravenously in hyperglycemic NOD mice and doxacyclin was injectedafter 5 days. Injection of PD-L1-transduced KLS reverted hyperglycemiain NOD mice (n=2) for 18.5±2.5 days (FIGS. 5A-5B). PD-L1⁺ KLS reducedCD4-restricted anti-BDC2.5 autoimmune response in vitro. We challengedCD4⁺ T cells extracted from splenocytes of 10-week-old NOD mice in ananti-BDC2.5 stimulation ELISPOT assay with the addition of autologousHSCs generated using the pharmacologic approach already described(ratios of 1:1, 1:10, 1:100 of HSCs to effector cells). However, adefect in HSC immunoregulatory properties was evident in NOD mice.

Example 4

Functional Human Studies

Autologous haematopoietic stem cell transplant (AHSCT, also known asbone marrow transplant) is an immunosuppressive chemotherapy treatmentcombined with reinfusion of blood stem cells to help re-build the immunesystem. AHSCT in new-onset T1D rendered normoglycemic nearly 60% oftreated individuals at 6 months. In a group of 65 individuals followedup for 48 months, AHSCT in a non-myeloablative setting achieved insulinindependence in nearly 60% of T1D individuals within the first 6 monthsafter receiving conditioning immunosuppression (ATG+Cyclophosphamide)and a single infusion of autologous HSCs. 32% of treated subjectsremained insulin-independent at the last time point of their follow-up.Treated subjects showed a decrease in HbA1c and an increase in C-peptidelevels as compared to pre-treatment.

Despite a complete immune recovery (i.e. leukocyte count) aftertreatment, 52% of treated individuals experienced adverse effects. HSCsof T1D individuals are defective in their immunoregulatory properties.We challenged CD4⁺ T cells extracted from PBMCs of healthy subjects orT1D individuals in an anti-CD3/-CD28 stimulation assay with the additionof autologous CD34⁺ cells (human HSCs) newly generated using ourpharmacologic approach (1:1, 1:10, 1:100 ratio of HSCs to effectorcells). Addition of HSCs obtained from healthy subjects led to adose-dependent decrease of IFN-γ-producing CD4+ T cells. On thecontrary, a defect in immunoregulatory properties was evident when HSCsfrom individuals with T1D were added. HSCs exhibited impairedmobilization in individuals with T1D. To confirm that the mobilizationof HSCs cells (CD34+) is not an expression, we evaluated themobilization properties of HSCs in T1D individuals. We thus establisheda trial (NCT01102699) that was performed with Padua University (Dr.Gianpaolo Fadini), in which we tested bone marrow responsiveness to 5μg/kg hrG-CSF in 6 individuals with T1D. While CD34⁺ cells significantlyincreased in healthy controls, an impaired mobilization of CD34+ wasobserved in T1D individuals. This data confirm the existence of a HSC“mobilopathy” in T1D individuals. HSC mobilization with a CXCR4antagonist does not increase PD-L1⁺ HSCs of T1D individuals. To assesswhether mobilization alters expression of PD-L1 on HSCs we studied theimmune phenotype of HSCs before and after mobilization with anti-CXCR4in 5 healthy subjects and 8 individuals with T1D. While PD-L1 expressionon CD34⁺ cells increased in healthy subjects after mobilization (6.2±0.6vs. 0.6±0.2, p=0.0001), it did not change in CD34⁺ cells of T1Dindividuals (4.7±1.5 vs. 1.5±0.8) highlighting that CD34+ cells requirean in vitro manipulation to overturn PD-L1 defect and recover theirimmunoregulatory properties.

Example 5

PGE₂ Highly Augment PDL1 Expression in HSC Cells Both Murine and Human.

Murine: we cultured isolated HSCs (KL cells) in a serum-free culturemedium supplemented with standard stem cell growth factors and pulsedwith the novel small molecule derived from prostaglandins E2 (PGE₂) atdifferent timepoints during a 8-day culture. Briefly, peripheralLin^(neg)Sca-1⁺Kit⁺ cells were isolated from 10-week-old NOD mice, and150-200 plated into each well on a 96-well plate with 200 ml of Stemspanserum-free medium (Stem-Cell Technologies) supplemented as alreadydescribed. PGE₂, which has been shown to implement expansion ofmurine/human isolated HSCs in vitro and it is now being tested in humansin phase II clinical trials, has been added (2 μl, 10 μM, Chemicon) at24 h, 96 h and at 6 days to enrich the pool of PD-L1⁺ HSCs newlygenerated. Cells were cultured for 8 days at 37° C. in 5% CO₂.

Human: human HSCs were cultured using StemSpan supplemented with humanstem cell growth factors as previously reported (22). Briefly, CD34⁺cells were plated at 5×10⁵ cells/ml in supplemented StemSpan on a96-well plate, at 200 μl/well for 7 days. HSCs were pulsed with PGE₂ asdescribed in murine experiments. PD-L1⁺ HSCs were quantified by FACSanalysis at different timepoints and at the end of the procedure.

Example 6

Treatment Protocol with PD-L1⁺ Expressing Hematopoietic Stem Cell.

Initial evaluation—Patients will undergo standard work-up for autologousbone marrow transplantation according to institutional guidelines, andthen undergo two bone marrow harvests at a minimum of 4 weeks apart thatwill be used for a back-up marrow (minimum of 2×10⁶ CD34⁺ cells/kg) andfor a harvest of autologous bone marrow (target of 5×10⁶ CD34⁺ cells/kgwith a minimum of 4×10⁶ CD34⁺ cells/kg).

Harvest of a back-up autologous graft—Hematopoietic cells will becollected from the patient in advance of the treatment, to serve as asalvage procedure (“back-up graft”), should there be no hematopoieticrecovery observed 6 weeks following the injection ofgenetically-manipulated cells, or should manipulated cells fail to meetrelease criteria. Bone marrow (up to 20 ml/kg) will be harvested fromthe patient under general anesthesia from the posterior iliac crests onboth sides by multiple punctures at a minimum of 4 weeks prior to genetherapy. A portion of the bone marrow containing 2×10⁶ CD34+ cells/kgwill be frozen and stored unmanipulated in liquid nitrogen vapors (−162°C. and −180° C.) according to standard clinical procedures forautologous bone marrow collection to constitute the back-up graft. Theremainder of the harvest will be selected for CD34+ cells (describedbelow) and utilized for gene modification (described below).

Bone marrow harvest—The remainder of the first bone marrow harvest inexcess of the needed back up marrow will be utilized with a second bonemarrow harvest for gene transfer. The second harvest will occur nosooner than 4 weeks after the initial harvest (described above). For thesecond harvest, bone marrow will again be harvested from the patientunder general anesthesia from the posterior iliac crests on both sitesby multiple punctures. The amount of marrow collected will be up to 20ml/kg of body weight. This will give a total nucleated cell count ofgreater than ˜4×10⁸ cells/kg. This in turn should yield a CD34⁺ celldose of greater than 4×10⁶ cells/kg after CD34+ cell selection.

Subjects from whom the estimated CD34+ count of both harvests is <4×10⁶cells/kg will not receive conditioning. After a period of at least 6weeks, if the subject wishes to remain on study, he may be harvestedagain. Subjects withdrawn from the study prior to administration oftransduced CD34+ cells will resume normal clinical care (supportive careand/or allogeneic HSCT). Efficacy and safety assessments will not becarried out from the point of withdrawal and data will not be collectedfor the database.

CD34+ cell purification—To allow sufficient time for clearance ofconditioning agents and minimize the time of pre-stimulation andculture, whole bone marrow will be held overnight. The bone marrow willbe red cell-depleted by density gradient centrifugation. CD34+ cellswill be positively selected from the bone marrow mononuclear cells usingthe CliniMACS reagent and instrument. Quality control (QC) samples aretaken to assess purity and sterility. Purified cells will be immediatelyprocessed for pre-stimulation and transduction.

CD34+ cells pre-stimulation and transduction with vector—Transductionwill be carried out on one or both harvests. Transduction of cells inexcess of the back-up marrow target from the first harvest will betransduced and frozen for future use. The second harvest will be usedfor gene transfer in its entirety and the transduced product of thesecond harvest will be infused with the thawed transduced cells from thefirst harvest after conditioning.

Purified CD34+ cells are seeded in closed culture bags at a density of0.5-1×10⁶/ml in serum-free medium supplemented with growth factors(IL-3, SCF, FLT3L, TPO) and placed in an incubator at 37 C, 5% CO₂.After 24-30 hours, cells are harvested and counted. Additional QCtesting includes cell viability, and Colony Forming Unit (CFU) assay.Cells are transferred to a new culture bag and treated with lentiviralsupernatant. For this first round of transduction, cells are incubatedfor 18-24 hours. Cells are then harvested, counted, and transferred to anew bag, with lentiviral supernatant for a second round of transduction.

Final harvest and formulation—After the second round of transduction,cells are harvested, washed in plasmalyte and resuspended in their finalformulation (PLASMALYTE, 1% HSA) in a volume of 50-100 mL. All cellsavailable after removal of the QC samples will be infused into thepatient. QC includes cell count, viability, sterility on washsupernatant, Mycoplasma, Endotoxin on supernatant, phenotype, CFU, RCL(samples taken and archived), insertional analysis, and average vectorcopy number by qPCR (cultured cells). A sample for Gram stain is takenfrom the product immediately before delivery to the patient.

CD34+ cells ex vivo culture and stimulation with PGE₂—Purified CD34+cells are seeded in closed culture bags at a density of 0.5-1×10⁶/ml inSTFIA medium was defined as serum-free medium supplemented with 10 μg/mlheparin, 10 ng/ml human SCF, 20 ng/ml human TPO, 10 ng/ml human FGF-1,100 ng/ml IGFBP2, and 500 ng/ml Angptl3, placed in an incubator at 37 C,5% CO₂ and cultured for 48 h. The media is changed and PGE₂ is added tothe cells to achieve a final concentration of 0.1 μM. After another 24 hhrs, PGE₂ is added to the cell again. The cells are harvested at 1-8days after the second PGE₂ addition. For harvest later than day 2,addition PGE₂ in added to the culture media at day 2, day 4 and day 6,together changes of culture media.

Testing prior to subject re-infusion—Samples are collected during and atthe end of the procedure for cell count and viability (trypan blueexclusion or equivalent), sterility, mycoplasma, transduction efficiency(vector copy number), Gram stain, endotoxin and RCL testing. Of theseonly cell viability, sterility (in process, 72 hours), Gram stain andendotoxin measurements will be available prior to infusion.

If microbiological cultures reveal transient bacterial contamination, byGram stain or positive culture at 72 hours, Cell Manipulation CoreFacility staff will contact the PI, the assistant medical director andattending physician to decide whether to infuse the back-up harvest orinfuse the product with antibiotic coverage. If back-up harvest isinfused, the subject will be withdrawn from the protocol. If the cellviability is <70%, sterility testing is positive, or endotoxin is >5EU/kg/hr, the cells will not be returned, back-up harvest will beinfused and the subject will be withdrawn from the protocol.

If viable cell count from both harvests/transductions is greater than orequal to 4×10⁶ CD34+ cells/kg at the end of transduction, cells will beinfused. If viable cell count from both harvests/transductions is lessthan 4×10⁶ CD34+ cells/kg at the end of transduction, cells will not beinfused and back-up harvest will be infused 48 hours later.

Samples of the CD34+ cells may be tested for PD-L1 expression.

Subjects withdrawn from the study prior to administration of transducedCD34+ cells will resume normal clinical care (supportive care and/orallogeneic HSCT). Efficacy and safety assessments will not be carriedout from the point of withdrawal and data will not be recorded in theCase report forms (CRFs).

Subject conditioning regimen—Subjects will receive myeloablativeconditioning with Busulfan (˜4 mg/kg intravenously daily, adjusted forweight, (given over 3 hours once daily) administered on days −4 to −2,prior to infusion of transduced cells. Conditioning will occurconcurrent with purification and transduction of bone marrow cells.Busulfan levels will be drawn on all 3 days of administration, andlevels on days 1 and 2 will be used to adjust for weight.

Infusion of transduced cells—Cells will be infused intravenously over30-45 minutes after standard prehydration and premedication according toconventional hospital Hematopoietic Stem Cell Transplantation Unitstandard guidelines. This standard requires that the patient be oncontinuous cardiac, respiratory and oxygen saturation monitor throughoutthe infusion and for 30 minutes afterwards. Vital signs will be measuredand recorded pre-transfusion, 15 minutes into transfusion, every hourfor duration of infusion, and end of transfusion. The RN will stay withthe patient for the first 5 minutes of the transfusion. If twotransduction products are administered, the second transduced productwill be administered without delay after the first.

Example 7

ToleraCyte™, a programmed CD34⁺/PD-L1⁺ immuno-regulatory cell product ofFate Therapeutics, Inc., have been show to treat T1D mice. ToleraCyte™is a programmed CD34+ cell immunotherapy that is undergoing preclinicalinvestigation for the treatment of autoimmune and inflammatorydisorders. The immuno-regulatory cell therapy is comprised of CD34+cells that have been programmed ex vivo with a proprietary combinationof pharmacologic modulators. ToleraCyte is designed to optimize thecapacity of CD34+ cells to effectively traffic to sites of inflammationand express potent T-cell regulatory factors, including PD-L1 and IDO1.

In preclinical experiments on well-established non-obese diabetic (NOD)mice, the mouse model of human Type 1 diabetes (T1D), a singleadministration of programmed cells ToleraCyte™ results in durablecorrection of T1D diabetes in a NOD mouse model. The hyperglycemic NODmice are designed to mimic new-onset type 1 diabetes. In addition, itwas also shown that in pre-hyperglycemic NOD mice, a singleadministration of programmed cells ToleraCyte™ statistically andsignificantly delays the onset of T1D in NOD mice, where the median timeto onset was not reached by Day 140 as compared to untreated mice(median time to onset=Day 115; p=0.0004).

Furthermore, in a humanized model of type 1 diabetes, programmed CD34⁺cells showed enhanced trafficking to the pancreas and regulation ofT-cell activation. Together, these preclinical results support thepremise that ToleraCyte™ can serve as a disease-modifying immunotherapyfor patients with type 1 diabetes. (SAN DIEGO, Jun. 11, 2016 (GLOBENEWSWIRE).

Example 8

Experimental Design and Methods

Design and Methods for Human Studies

Patients Characteristics—Blood samples were obtained from new onsetdiabetic individuals (New-onset T1D), long-standing diabetic individuals(T1D) and healthy individuals (CTRL) in accordance with The San RaffaeleScientific Research Institute under an Institutional Review Boardcommittee approval. Peripheral blood mononuclear cells (PBMC) fractionswere isolated by Ficoll density gradient centrifugation for cellculturing experiments. Additional blood samples were obtained from T1Dsubjects and CTRL at baseline before treatment and 6 hours aftertreatment with CXCR4-antagonist (Mozobil; Sanofi) at the dose of 0.24mg/kg body weight in accordance with the Institution Review BoardCommittee of Padova (2996P) and was performed in accordance with theDeclaration of Helsinki (Clinical trial registered on clinicaltrials.gov(NCT02056210)). Patients with Type 1 Diabetes aged 18-65 years wererecruited among those referred to the diabetes outpatient clinic of theUniversity Hospital of Padova. Individuals without diabetes aged 18-65years were recruited from those referred to the same outpatient clinicfor screening of other metabolic diseases. All provided written informedconsent. Exclusion criteria were pregnancy or lactation; recent (within2 months from study entry) surgery, trauma, or acute diseases; immunediseases (except from type 1 diabetes and autoimmune thyroiditis);chronic infectious diseases; hematologic malignancies either past orpresent; solid tumor known or strongly suspected; leukocytosis,leukopenia, or thrombocytopenia; solid organ transplant orimmunosuppression; alteration of hepatic function (transaminases >2upper limit of normality); severe chronic diabetic micro- ormacroangiopathy; HbA_(1c)>11%; deficit in renal function (estimatedglomerular filtration rate <50 mL/min/1.73 m²); significantabnormalities of the peripheral lymphocyte immunophenotype; knownhypersensitivity to plerixafor or its excipients; and refusal orinability to provide informed consent. Women with childbearing potentialcould participate in the study if on oral contraception, and a negativepregnancy test was required before study entry. Women were also asked tocontinue oral contraception for 3 months after plerixaforadministration. All medications for the treatment of diabetes and forother medical conditions were allowed during the study.

Human antibodies—The following antibodies were used for flow cytometricanalysis in the reported studies: phycoerythrin (PE)-conjugatedanti-human PD-L1 (CD274) or allophycocyanin (APC)-labeled anti-humanPD-L1 (CD274), PE-conjugated anti-human PD-1 (CD279), PE-conjugatedanti-human PD-L2 (CD273), PE-conjugated or R-Phycoerythrincyanin 5.1(PC5) conjugated anti-human CD34, fluorescein isothiocyanate(FITC)-conjugated anti-human CD45, PE-conjugated anti-human-CD19,peridin-chlorophyll-protein complex (PerCP)-conjugated anti-human CD11cand Pacific Blue (PB)-conjugated anti-human CD16 were purchased from BDBiosciences, Biolegend or Beckman Coulter. The following antibodiescorresponded to different isotype controls for the abovementioned humanantibodies: PE-conjugated mouse IgG1K, mouse PC5-conjugated IgG1,APC-labeled mouse IgG2bκ.

Human flow cytometric analysis—To assess PD-L1, PD-L2 and PD-1expression on human HSCs, fresh blood collected from healthyindividuals, T1D and new-onset T1D individuals was stained with PE-Cy5.5anti-human CD34, PE anti-human PD-L1 or PD-L2 or PD-1 (BD Biosciences).Fresh blood was also stained with PE anti-human PD-L1 together withPECy7 anti-human-CD19, APC anti-human CD11c or Pacific blue anti-humanCD16 (all BD Biosciences) to assess PD-L1 expression on B cells,dendritic cells or monocytes, respectively. BD LSRFortessa flowcytometer (BD Biosciences) was used to analyze cells with the lightscatter properties of stem cells or lymphocytes. Background staining wasdetermined using nonreactive isotype-matched control mAbs with gatespositioned to exclude 99% of non-reactive cells. FlowJo software version8.7.3 (Treestar, Ashland, OR) was used for analysis. Apoptosis wasassessed by permeabilization of previously isolated CD34+ cells, whichwere next stained with APC Annexin V (BD Bioscience) while dead cellswere detected using a Fixable Viability Dye Staining (Amcyan,eBioscience).

In vitro proliferation assay and glucose challenge of human CD34+cells—CD34+ cells were first isolated using magnetic beads (Miltenyikit) from PBMCs obtained from blood samples of enrolled subjects. Next,CD34+ cells were stained with CFSE (FITC, Invitrogen C1157) and culturedfor 72 hours at 37° C. in 5% CO2 in StemSpam SFEM II media (StemCellTechnologies). Proliferation was visualized by flow cytometry accordingto the dye dilution at 24 h, 48 h and 72 h. To assess whether glucoseexposure affects PDL-1 expression on CD34+ cells, we cultured CD34+cells, previously isolated from PBMCs obtained from CTRL and T1D, inDMEM without serum at different glucose concentrations (5 mM, 20 mM and35 mM) for 72 h. PDL-1 expression was assessed by FACS as previouslydescribed.

Pharmacological modulation of human HSCs CD34+ HSCs—1×10⁶ of isolatedhuman CD34+ HSCs cells were cultured in 200111 of StemSpan SFEM II mediasupplemented with recombinant human SCF (50 ng/ml), recombinant humanTPO (50 ng/ml), recombinant human FLT3-L (50 ng/ml), human IFN-β (1000U/ml), human IFN-γ (5 ng/ml) and human Polyinosinic-polycytidylic acid(Poly I:C) (1 μg/ml) in a U-bottom 96-well plate at 37° C. in 5% CO₂.PD-L1 expression was evaluated before and after 24 hours of culture byflow cytometry using anti-human CD34 and anti-human PD-L1, and withtheir corresponding isotype controls.

Human ELISpot assay—An ELISPOT assay was used to measure the number ofIFN-γ-producing cells according to the manufacturer's protocol (BDBiosciences, San Jose, CA) as previously showed by our group (16). 1×10⁶PBMC, isolated from T1D patients, were cultured for 48 h in presence ofIA-2 (100 μg/ml) peptide in RPMI media supplemented with 10% FBS. At dayone after stimulation, 500 μl of media were added to the culture. Cellswere collected at day 2 and plated in a human IFN-γ ELISpot assay withor without CD34+ HSCs, Trifecta-modulated CD34+ HSCs in a ratio of 1:1,1:2, 1:4, or 1:8 in RPMI media un-supplemented. Spots were counted usingan A.El.VIS Elispot Reader (A.EL.VIS GmbH, Hannover, Germany) or on anImmunospot Reader.

Western blot—Total proteins of intestinal bioptic samples were extractedin Laemmli buffer (Tris-HCl 62.5 mmol/l, pH 6.8, 20% glycerol, 2% SDS,5% β-mercaptoethanol) and their concentration was measured (Lowry etal., 1951). 35 μg of total protein was electrophoresed on 7% SDS-PAGEgels and blotted onto nitrocellulose (Schleicher & Schuell, Dassel,Germany). Blots were then stained with Ponceau S. Membranes were blockedfor 1 h in TBS (Tris [10 mmol/l], NaCl [150 mmol/l]), 0.1% Tween-20, 5%non-fat dry milk, pH 7.4 at 25° C., incubated for 12 h with a polyclonalgoat anti-human Pdcd-1L1 antibody (Santa Cruz Biotechnology, Santa Cruz,CA, USA) diluted 1:200 or with a monoclonal mouse anti-β-actin antibody(Santa Cruz Biotechnology) diluted 1:1000 in TBS-5% milk at 4° C.,washed four times with TBS-0.1% TWEEN®-20, then incubated with aperoxidase-labeled mouse anti-goat IgG secondary antibody (or rabbitanti mouse for β-actin) diluted 1:1000 (Santa Cruz Biotechnology) inTBS-5% milk, and finally washed with TBS-0.1% Tween-20. The resultingbands were visualized using enhanced chemiluminescence (SuperSignal;Pierce, Rockford, IL, USA).

qRT-PCR—RNA from isolated CD34+ cells was extracted using Trizol®Reagent (Invitrogen), and qRT-PCR analysis was performed using TaqManassays (Life Technologies, Grand Island, NY) according to themanufacturer's instructions. The normalized expression values weredetermined using the ΔCt method. Quantitative reverse transcriptasepolymerase chain reaction (qRT-PCR) data were normalized for theexpression of ACTB, and ΔCt values were calculated. Statistical analysiscompared gene expression across all cell populations for each patientvia one-way ANOVA followed by Bonferroni post-test for multiplecomparisons between the population of interest and all otherpopulations. Statistical analysis was performed also by using thesoftware available RT² profiler PCR Array Data Analysis (Qiagen). Fortwo groups comparison Student t test was employed. Analysis wasperformed in triplicates after isolation of fresh CD34⁺ cells. Below arereported the main characteristics of primers used:

Gene UniGene Refseq Band Reference Symbol # Accession # Size (bp)Position DC274 Hs.521989 NM_001267706.1 89 614 (PDL-1)

Confocal microscopy—Bone marrow sections from Type 1 diabeticindividuals and from healthy control subjects and then stained with thecorresponding antibodies. Images were captured on Zeiss LSM 510 Metaconfocal microscope (Carl Zeiss SpA). Details of the staining procedurecan be found in supplemental procedures.

Design and Methods for Murine Studies

Animals—Female NOD/ShiLtJ (NOD) and male C57BL/6J mice were purchasedfrom The Jackson Laboratory (Bar Harbor, ME). All mice were usedaccording to institutional guidelines, and animal protocols wereapproved by the Boston Children's Hospital Institutional Animal Care andUse Committee.

Diabetes Monitoring—Overt diabetes was defined as blood glucose levelsabove 250 mg/dL for 2 consecutive days. Blood glucose was measured usingthe Breeze2 (Bayer S.p.A., Viale Certosa, Milano, Italy) blood glucosemeter.

Reversal studies—Female NOD mice were monitored beginning at 10 weeks ofage, and on day 2 of hyperglycemia (>250 mg/dl), were injected withKL-PD-L1.Tg cells, or HSCs-unmodulated cells, HSCs-mock-transduced cellsor HSCs-modulated with trifecta (see description above), wereadministered as 3×10⁶ cells via vein tail. Mice were monitored daily bymeasuring blood glucose until the time of sacrifice (normoglycemia wasobserved the following day post-treatment as below 250 mg/dl), andmeasurements were performed by tail bleeding according to NationalInstitutes of Health guidelines. Bone marrow cells were obtained fromfemurs and tibiae of NOD and C57BL/6J mice by flushing with phosphatebuffered saline (PBS). Bone marrow cells were lineage depleted by usingthe Lineage Negative Depletion Kit (Miltenyi Biotec). Upon depletion,lineage negative c-kit⁺ cells were isolated using CD117 Microbeads(Miltenyi Biotec), following the manufacturer's instruction.

Murine flow cytometry antibodies—The following antibodies were used forflow cytometric analysis for assessing phenotypic characterization of KLextracted from bone marrow and spleen: phycoerythrin (PE)-conjugatedanti-human PD-L1 (CD274) or allophycocyanin (APC)-conjugated ratanti-mouse PD-L1 (CD274), phycoerythrin (PE)-conjugated rat anti-mousePD-1 (CD279), phycoerythrin (PE)-conjugated rat anti-mouse PD-L2(CD273), were purchased from BD Biosciences or Biolegend respectively.The following antibodies corresponded to different isotype controls forthe abovementioned murine antibodies: PE Mouse IgG1, κ Isotype Ctrl; andAPC Mouse IgG2b, κ Isotype Ctrl.

Immunophenotypic characterization of murine KL characterization—MurineKL cells previously extracted from bone marrow were suspended in 2004,of buffer, then stained with the following antibodies and incubatedaccording to manufacturer's instructions for 30 minutes at 4° C. Cellswere washed with buffer, centrifuged at 300 g for 10 minutes andsuspended in 300 μl of buffer. The following antibodies were used forthe staining: Rat anti-mouse CD274 or CD273 or anti-mouse CD279. PD-L1,PD-L2 and PD-1 expression on KL cells was represented as histograms.

Extracted bone marrow from 8 weeks NODs and B6 mice were subject to redblood lysis with ACK-lysing buffer (BD lysis buffer) followed by awashing step in Flow buffer (BD staining buffer). Bone marrow cells werestained with the following cocktail of antibodies:

Anti-Lineage negative cocktail-APC, anti-C-kit-PerCP, anti-Sca1-FITC,anti-CD150-PE, anti CD41FITC, anti-CD48-PerCP, anti-CD244 PerCP,anti-PD-L1-PE and anti-PD-L1-APC. All antibodies were purchased fromeBioscience and from BD Pharmingen. Samples will be incubated for 30 minin the dark at 4° C. and then washed again with Flow Medium andeventually fixed with Formalin 1%. Samples will be examined at FACSCalibur and results will be analysed using Flowjo software.

Flow cytometric analysis, Non-hematopoietic stem cellscharacterization—1×10⁶ cells per sample will be stained with anti-mouseB220-PE to assess B cells and dendritic cells will be determined withanti CD11c-PerCP and monocytes with anti-mouse F4/80 APC. PD-L1expression in B220⁺ cells, CD11c⁺ cells and CD16⁺ cells was assessed byusing anti-mouse CD274-PE. Briefly, isolated bone marrow cells andsplenocytes were washed in Flow Medium (PBS with 2% of FCS and 0.05% ofsodium azide) and stained with the appropriate dilution of flowantibodies. Samples will be incubated for 30 min in the dark at 4° C.and then washed again with Flow Medium and eventually fixed withFormalin 1%. Samples will be examined at FACS Calibur and results willbe analyzed using Flowjo software. anti-mouse CD11c-PerCP was purchasedfrom Biolegend, anti-mouse F4/80 APC from eBioscience and anti-mousePD-L1 used is from BD Pharmingen.

Apoptosis assay—Isolated KL cells were washed twice with cold PBS andthen resuspended in 1× Binding Buffer (component no. 51-66121E; BDPharmingen) at a concentration of 1×10⁶ cells/ml. Then 100 μl of thesolution (containing 1×10⁵ HSCs) were transferred into a 5 ml culturetube and proceeded by a staining with 5 μl of PE Annexin V and 5 μl7-AAD and followed by a vortex of cells and incubation for 15 min at RT(25° C.) in the dark. After incubation, 400 μl of 1× Binding Buffer toeach tube were added to each tube prior to their acquisition by flowcytometry. The following controls were used to set up compensation andquadrants: unstained cells, cells stained with PE Annexin V (no 7-AAD)and cells stained with 7-AAD (no PE Annexin V). Cells that stainedpositive for PE Annexin V and negative for 7-AAD were undergoingapoptosis. Cells that stained positive for both PE Annexin V and 7-AADwere either in the end stage of apoptosis, were undergoing necrosis, orwere already dead. Cells that stained negative for both PE Annexin V and7-AAD were alive and not undergoing measurable apoptosis.

In vitro proliferation assay—Isolated KL cells were washed twice withcold PBS buffer without FCS, then resuspended in half final volume ofbuffer at 3×10⁷ cells/ml and into the other half of volume was addedCFSE to reach a final concentration of 10 uM. Diluted CFSE will be addedto cell suspension followed by a vortex and incubation at 37° C. for 15minutes. After incubation, FCS was added to cell suspension in order toquench any remaining free CFSE, and the tube will be filled completelywith PBS buffer. After a second wash, cells were resuspended in mediaand were cultured for 3 days at 37° C. in 5% CO₂. After 72 hproliferation of KL cells can be visualized at flow cytometry accordingto the dye dilution.

Murine ELISpot assay—An ELISPOT assay was used to measure the number ofIFN-γ-producing cells according to the manufacturer's protocol (BDBiosciences, San Jose, CA) as previously showed by our group (16). 1×10⁶of splenocytes, isolated from NOD-treated mice (NOD-PD-L1.Tg treated,NOD-Trifecta-treated, NOD-KL-treated) and NOD-untreated mice, werecultured for 24 hours in presence of the following murine isletspeptides (150 μg/ml): BDC2.5, IGRP, GAD65 and insulin at 300 μg/ml.Spots were counted using an A.El.VIS Elispot Reader (A.EL.VIS GmbH,Hannover, Germany)

Cell Lines and Cell Culture—Lenti-XTM 293T Cell Line used in this studywas purchased from Clontech as recommended. All procedures involvinghuman cell line HEK293T and lentiviral methodologies were approved bythe Institutional Biosafety Committee (IBC) of Boston Children'sHospital Committee, Harvard Medical School.

Lentivirus Production and Transduction—The full-length cDNA encodingmurine PD-L1 was cloned into the transfer plasmidpHAGE-fullEF1α-MCS-IZsGreen. VSV-G pseudotyped lentiviruses weregenerated by co-transfection of the murine PD-L1 transfer plasmidtogether with the packaging expression plasmids (Gag/Pol, Tat, Rev) andthe envelope expressing plasmid encoding for VSV-G into 293T cells usingthe Trans-IT 293 transfection reagent (Mirus). 24 or 48 hours posttransfection, the supernatant containing the viral particles wascollected, centrifuged at 1800 rpm for 5 minutes to remove dead cellsand debris, and concentrated using the Lenti-X concentrator followingmanufacturer's protocol (Takara Clonetech). Viral stocks were stored at−80° C. until transduction experiments were performed. Freshly isolatedmurine KL cells were transduced with recombinant PD-L1 lentiviralparticles in Stem SFEMII (Stem cell Technology) in presence of 2 μg/mLpolybrene (Sigma), 10 ng/ml of SCF and 100 ng/ml of TPO. 24 hours aftertransduction, cells were collected for FACS analysis and used forreversal studies.

Luciferase assay—KL cells isolated fromNOD.FVB-Tg(CAG-luc,-GFP)L2G85Chco/FathJ were purchased from JacksonLaboratory then were transduced with PD-L1 lentivirus and injected toNOD-hyperglycemic. After 24 hours, treated mice were injected withluciferin. Following luciferin injection, luciferase expression isassessed by IVIS Spectrum. Details of the whole procedure can be foundin supplemental methods.

Modulation of murine KL cells HSCs—Murine bone marrow KL cells wereisolated using magnetic beads and MACS® separation columns (Miltenyi)and ˜2×10⁵ cells were plated in a U-bottom 96-well plate (3799; Corning)with 200 μl of the following medium. Stemspan-SFEMII (StemCellTechnologies) supplemented with a cocktail of different growth factors.Cells were cultured for 24 hours at 37° C. in 5% CO₂. PD-L1 expressionwas evaluated before culture by FACS using rat anti-mouse PD-L1 (BDPharmingen) with the corresponding isotype control Rat IgG2a, λ (BDPharmingen).

Trifecta modulation—Isolated KL cells were resuspended in SFEMII(StemCell Technologies) supplemented with 50 ng/ml of recombinant humanSCF (StemCell Technology), 50 ng/ml of Mouse TPO (StemCell Technology),50 ng/ml of Recombinant Mouse IL-3 (R&D SYSTEMS), Recombinant MouseIFN-β (1000 U/ml) (R&D SYSTEMS), Mouse IFN-γ (5 ng/ml) (R&D SYSTEMS) and1 μg/ml of human Ploy (I:C) (Polyinosinic-polycytidylic acid)(InvivoGen).

Western blot—Murine KL cells were homogenized in RIPA buffer (20 mM TrispH 8.0, 150 mM NaCl, 0.1% SDS, 0.5% DOC, 0.5% triton X-100) withprotease inhibitors cocktail (Roche). Cell lysates equivalent to 50 μgof total protein were fractionated on 4%-20% SDS-polyacrylamide gradientgels (Bio-Rad) and transferred to nitrocellulose membranes (0.2 μm,Bio-Rad). Membranes were blocked with 5% BSA at room temperature for 1hour and then incubated overnight with anti-PD-L1 (Santa CruzBiotechnology), anti-Rabbit GAPDH (Cell Signaling TECHNOLOGY). Detectionwas performed by using anti-rabbit IgG, HRP-linked antibodies.

qRT-PCR—To measure expression levels of PD-L1 gene in KL cells. TotalRNA was extracted from KL cells and treated at 42° C. for 30 min with100 μl of extraction buffer (Arcturus Picopure, Applied Biosystems),then subject to different washing steps and eluted in 15 ul of elutionbuffer according to manufacturer's instruction. RNA was quantified bynano-drop spectrophotometer followed by reverse transcription andpre-amplification using ABI Reverse Transcription and Taqman PreAmp Kit(Applied Biosystems) according to the manufacturer's instruction. TaqMangene expression assays (Applied Biosystems) were performed on triplicatesamples using a 7900HT fast real-time PCR system (Applied Biosystems).Data were normalized relative to GAPDH house keeping gene.

Confocal microscopy and immunofluorescence of HSCs—Bone marrow extractedfrom femur and tibiae of 8 weeks old NOD and B6 mice were embedded inOCT and snap frozen in −80° C. N-methylbutane chilled in a slurry ofethanol and dry ice. Sections (7 μm) were prepared using a Microtome andair dried then stained with the corresponding antibodies. Images werecaptured on Zeiss LSM 510 Meta confocal microscope (Carl Zeiss SpA).Details of the staining procedure can be found in supplementalprocedures.

Murine GWAS assays—Methods for MTA 1.0 and HTA 2.0 AffymetrixMicroarray.

Genome-wide expression analysis was performed following AffymetrixGeneChip WT Pico protocol. RNA isolation was conducted using ArcturusPicoPure RNA Isolation Kit (Applied Biosystems) and then diluted toroughly 1.0 ng. RNA integrity was assessed for all RNA samples and thefinal concentration was measured on a Bioanalyzer using RNA Pico Chips(Agilent Technologies). Only RNA with a RIN score of 7 or higher wereused. Between 1-2 ng was used as template to construct cRNA through aseries of reactions involving cDNA synthesis, adaptor synthesis and a 16hr amplification step (Affymetrix). Following cRNA purification andquantisation, ss-cDNA was synthesized, fragmented and labeled(Affymetrix). Each MTA 1.0 or HTA 2.0 Genechip was hybridized for 17 hrsat 45 C. Arrays were then stained on a FS450 Fluidic station(Affymetrix) and scanned on a Gene Chip 7G Scanner (Affymetrix). Probeintensities were normalized according to a log scale robust multi-arrayanalysis (Expression Console-RMA, Affymetrix) method and normalizedintensities were plotted with Spotfire 6.0 (Perkin-Elmer).

Statistical Analysis—Unless otherwise indicated, all data are shown asmean±SEM. Statistical analysis was performed using the unpaired Studentt test. A two-sided value of P<0.05 was considered statisticallysignificant. The Kaplan-Meier curve with the Wilcoxon test was used toanalyze the development of diabetes in mice. Statistical analysis wasperformed using GraphPad Prism software (GraphPad Software, Inc., LaJolla, CA).

Results

PD-L1 is defective in HSCs from NOD mice—In order to identify anypotential immunoregulatory defects in hematopoietic stem cells (HSCs) inmice prone to autoimmune diabetes, we firstly performed a widetranscriptomic profiling for immunoregulatory, anti-inflammatory andcostimulatory molecules of murine HSCs from NOD mice and compared itwith those obtained from C57BL/6 mice. Sca-1⁺Lineage⁻c-kit⁺HSCs, (KLS)obtained from NOD showed at transcriptomic profiling a decreasedexpression of PD-L1 transcript as compared to those from HSCs obtainedfrom C57BL/6 mice (FIG. 8A). We next used a wide range of techniques inorder to confirm PD-L1 defect in NOD HSCs. First, we performed Westernblotting assays in order to determine the expression of PD-L1 in KLScells from NOD and compare it to C57BL/6 mice, we used GAPDH as internalcontrol (FIG. 8B). After quantification of the Western blotting assays,a decrease in PDL-1 relative expression was evident in NOD compared toC57BL/6 mice (FIG. 8C). PD-L1 mRNA expression AS measured by RT-PCR inKLS cells confirmed the reduced PD-L1 mRNA expression in NOD HSCs (FIG.8D). Fewer PD-L1⁺ cells and an overall reduced PD-L1 expression wasevident in normoglycemic NOD mice in different bone marrow progenitors,namely in Sca-1⁺Lineage⁻c-kit⁺HSCs (KLS) cells, in Lineage⁻c-kit⁺ (KL)cells, in long-term repopulating HSCs (CD41⁻CD48⁻CD150⁺ cells) and(CD244⁻CD48⁻CD150⁺) as compared to C57BL/6 mice. (FIGS. 8F-8L).Interestingly, PD-L1 defect was mainly restrained to the HSC populationsin NOD mice, as other bone marrow-derived immune relevant cells (e.g.;B220⁺ B lymphocytes cells, CD11c⁺ dendritic cells and F4/80+macrophages) were not defective in PD-L1 (data not shown). Othercostimulatory molecules (e.g.; PD-L2, PD-1, CD40, CD80 and CD80) wereevaluated as well, and we did notice any significant difference betweenNOD and C57BL/6 HSCs obtained from bone marrow or spleen (data notshown), suggesting the unicity of PD-L1 defect. We sought then toexplore any association of PD-L1 defect with age or disease status, andthus we performed flow cytometry analysis on bone marrow and splenocytesextracted-KL cells of NOD and C57BL/6 mice, respectively at 4 weeks, 10weeks and above 16 weeks. We noticed a slight decline in the number ofPD-L1+ cells in both strain. We then aimed at understanding therelevance of PD-L1 within the HSC niche, and thus analyzed with confocalimaging bone marrow tissue from NOD and C57BL/6, determining that PD-L1is defective in HSCs in NOD mice (data not shown). In order to assess ifhyperglycemia may play a role in this defect or if a high HSC turnoverwith increased apoptosis may generate immature HSCs in NOD mice, wetested the effect of high glucose on PD-L1 expression in HSCs from NODand C57BL/6 mice and quantify their turnover and apoptotic rate.Isolated KL cells from NOD and C57BL/6 mice were cultured for 3 days inhigh glucose (20 and 35 mM)). While some changes were evident, noparticular pattern suggested the existence of any potential highglucose-associated effect on PD-L1 expression (data not shown). Then, weperformed a proliferation assay on CFSE labelled-HSCs from NOD and fromC57BL/6 mice at baseline, when cultured for 24 hours and when culturedfor 72 hours in SFEMII media. No differences in the proliferation ratewere evident among HSCs from NOD or C57BL/6 (data not shown). We havefurther studied the apoptotic rate of HSCs from NOD and from C57BL/6mice at baseline, after 24 hours of culture and after 72 hours ofculture. Although at baseline, HSCs from NOD displayed a higherpercentage of AnnexinV+/7-AAD− apoptotic cells as compared with HSCsfrom C57BL/6, after 24 hours and 72 hours of culture, an oppositescenario was evident with more apoptotic HSCs in C57BL/6 as compared toHSCs from NOD (data not shown). Our data confirmed the existence of aHSC-specific defect in PD-L1 expression in NOD mice, mainly restrainedto hematopoietic stem cells populations.

Genetically engineered NOD HSCs abrogate autoimmune response in vitro—Wetested the effect of a genetic-based engineering approach to overcomePD-L1 defect in NOD HSCs. We genetic engineered ex vivo murine KL cellsand generate PD-L1⁺.Tg HSCs from NOD mice by a third-generationself-inactivating lentiviral vectors (LV), which has a strong potentialuse in vivo because of its high efficiency and low risk of genotoxicity(Kevin D. Bunting and Cheng-Kui Qu, 2014, Methods in Molecular Biology,1185, DOI 10.1007/978-1-4939-1133-2_21) and explore their effect on theonset of experimental autoimmune diabetes in NOD mice. Isolated murineHSCs (KL) were transduced with PD-L1 pseudoviral particles previouslyobtained by infecting HEK 293TN producer cells with a lentivirus vectorcontaining PD-L1 gene whose expression was under the control of adoxycycline promoter, and a fluorescent marker designed as ZsGreen. Wethus successfully generated PD-L1⁺.Tg HSCs with an efficiency of 60%positive PD-L1⁺ cells as compared to nearly 7% pre-transduction (FIGS.9A-9C). An increased MFI was evident as well (pre-transduction=5.6±1.9;post-transduction=47.8±4.8). Immunofluorescence nicely depicted theincreased surface PD-L1 expression after transduction with PD-L1 LV(data not shown). Genome wide analysis of the newly generated PD-L1⁺.TgHSCs confirmed the PD-L1 upregulation of PD-L1 by nearly a 327-foldincrease compared to the Mock-LV transduced HSCs (data not shown). Wethen explored the immunoregulatory properties of newly generatedPD-L1⁺.Tg HSCs in an autoimmune setting in vitro. PD-L1⁺Tg.HSCsgenerated from normoglycemic NOD mice were cocultured at 3 differentratios to CD4+ CD25⁻ T cells (1:1; 1:5 and 1:10) with CD11c⁺ DCs andBDC2.5 transgenic CD4⁺CD25− T cells in the presence of the isletmimotope peptide BDC2.5. IFN-γ⁺ CD4⁺CD25⁻ T cells, as quantified by flowcytometry, showed a significant decrease when coculture with PD-L1⁺.TgHSCs at high ratio (p<0.005) compared with non transduced HSCs (KLcells) (FIGS. 9D and 9E). When PD-L1⁺.Tg HSCs were pre-cultured with ananti-PD-L1 blocking mAb, the aforementioned immunoregulatory effect wasseverely hampered (data not shown). The PD-L1 dependent immunoregulatoryproperties were confirmed by using the CD8-dependent assay wherePD-L1⁺.Tg HSCs were cocultured at 3 different ratios (1:1; 1:5 and 1:10)with CD11c⁺ DCs and 8.3 NOD transgenic CD8⁺ T cells in the presence ofthe islet mimotope peptide IGRP (data not shown). We then tested theimmunoregulatory effects of PD-L1⁺.Tg HSCs in a non autoimmune specificassay. CD4⁺ CD25⁻ T cells extracted from NOD normoglycemic werestimulated by soluble anti-CD3/anti-CD28 and cocultured with PD-L1⁺.TgHSCs at 3 different ratios to CD4⁺ CD25⁻ T cells (1:1; 1:5 and 1:10).The immunoregulatory effect was confirmed with a significant decrease inthe percentage of IFN-γ⁺ CD4⁺ CD25⁻ T cells when PD-L1⁺.Tg HSCs wereadded, although less evident as compared to the autoimmune assay, butstill PD-L1 dependent (FIGS. 9F and 9G).

Genetically engineered NOD HSCs reverted hyperglycemia—In order toevaluate the immunoregulatory properties in vivo of the newly generatedPD-L1⁺.Tg HSCs, newly hyperglycemic NOD mice were adoptively transferredwith 3×10⁶ PD-L1⁺.Tg HSCs (FIG. 9I) or 3×10⁶ non-transduced(un-manipulated) HSCs (FIG. 9K) respectively. PD-L1⁺.Tg HSCssuccessfully reverted hyperglycemia in 100% of treated hyperglycemic NODmice with 20% of treated mice remained normoglycemic till the completionof the study, while none of untreated hyperglycemic NOD mice (FIG. 9H)or of hyperglycemic NOD mice treated with doxycycline (FIG. 9J) revertedto normoglycemia. When untransduced HSCs were used, 1 hyperglycemic NODmouse reverted to normoglycemia and 1 showed a mild transientimprovement of glycemic levels, (data not shown). The pancreasimmuno-histopathology of PD-L1⁺.Tg HSCs-treated hyperglycemic NOD micerevealed no evidence of infiltration of the islets or mild lymphocyteinfiltration (data not shown), with preserved insulin staining ascompared to hyperglycemic untreated NOD, showing a better reducedinsulitis score. Immunophenotype of PD-L1⁺.Tg HSCs-treated hyperglycemicNOD-mice showed at day 14 after treatment a two fold increase in thepercentage of FoxP3⁺ regulatory CD4⁺ T cells as compared to untreatedmice, while no changes were observed in the percentage of IFN-γ⁺ andIL-17⁺CD4⁺/CD8⁺ T cells (data not shown). Quantification ofIFN-γ-producing cells in an ex-vivo assay of splenocytes challenged withislets peptides at day 40 (BDC2.5, IGRP, GAD-65 and insulin) revealed areduction of IFN-γ⁺ cells in PD-L1⁺.Tg HSCs-treated hyperglycemicNOD-mice as compared to untreated (data not shown).

Genetically engineered HSCs traffic to the pancreas in hyperglycemic NODmice—To explore the fate of infused PD-L1⁺.Tg HSCs in NOD mice, weperformed a set of tracking experiments in the pancreas, the spleen,pancreatic draining lymph node (PLN) and bone marrow by using the GFPtracer designed as ZsGreen within PD-L1⁺.Tg HSCs. PD-L1⁺.Tg HSCs wereadoptively transferred into normoglycemic and hyperglycemic NOD mice andtissues were harvested after 1 day, 7 days and 14 days from infusion.GFP⁺ cells and GFP (ZsGreen mRNA) were quantified in all tissues by flowcytometry and RT-PCR respectively. PD-L1⁺.Tg HSCs once infused intohyperglycemic NOD preferentially traffic to the pancreas (data notshown) and home to a lower extent to the spleen and PLN (data notshown). While, PD-L1⁺.Tg HSCs preferentially home to bone marrow intonormoglycemic NOD (data not shown). GFP⁺ cells were visualized byconfocal imaging into the pancreas of PD-L1⁺.Tg HSCs treatedhyperglycemic, but not normoglycemic, NOD mice NOD (data not shown).Luminescence images of NOD-hyperglycemic adoptively transferred withLuciferase⁺PD-L1.Tg KL cells within 24 hours of treatment furtherconfirmed our data. In conclusion, we hereby confirmed a substantialhoming of PD-L1⁺ HSCs to the pancreas in hyperglycemic NOD. We can nowpropose a working hypothesis, in which PD-L1⁺.Tg HSCs traffic into thepancreas and delete via PD-L1 dependent mechanism effector autoimmune Tcells.

Pharmacologically modulated HSCs abrogate autoimmune response invitro—The use of a genetic approach to cure T1D might not be an easytask, we explore the feasibility of a PD-L1 pharmacological modulationby small molecules. We tested the ability of single agents and of acocktail of agents to upregulate PD-L1. We came out with a cocktail of 3agents (that we named Trifecta: IFN-γ, IFN-β, Poly I:C) capable ofstrongly upregulating PD-L1, (from nearly 6% of PD-L1⁺ cells in apopulation of HSCs up to 65% of PD-L1⁺ cells in the population aftertreatment with Trifecta) and creating programmed HSCs (pHSCs).Immunofluorescence nicely depicted the increased PD-L1 surfaceexpression after modulation with small molecules, a combination ofgrowth factors (SCF, TPO, IL-3, IL-6, IFN-B, IFN-g and poly I:C) (datanot shown). Genome wide analysis confirmed the upregulation of PD-L1 inpHSCs with nearly a 13-fold increase compared to the unmodulated HSCs(data not shown). We then explored the immunoregulatory properties ofpHSCs in an autoimmune setting. pHSCs generated from normoglycemic NODmice were cocultured at 3 different ratios to CD4⁺ CD25− T cells (1:1;1:5 and 1:10) with CD11c⁺ DCs and BDC2.5 transgenic CD4+ CD25− T cellsin the presence of BDC2.5 peptides. The quantification by flow cytometryof IFN-γ⁺ CD4⁺ CD25⁻ T cells revealed a pronounced and significantdecrease when pHSCs were added (p<0.005) compared to controls (data notshown). When pHSCs were pre cultured with an anti-PD-L1 blocking mAb theimmunoregulatory effect was hampered (data not shown). The PD-L1dependent immunoregulatory properties were confirmed by using theCD8-dependent assay where pHSCs were cocultured at 3 different ratios(1:1; 1:5 and 1:10) with CD11c⁺ DCs and 8.3 NOD transgenic CD8⁺ T cellsin the presence of the islet mimotope peptide IGRP. We then tested theimmunoregulatory effects of pHSCs in a non autoimmune specific assay.CD4⁺ CD25⁻ T cells extracted from NOD normoglycemic were stimulated bysoluble anti-CD3/anti-CD28 and cocultured with pHSCs at 3 differentratios to CD4⁺ CD25⁻ T cells (1:1; 1:5 and 1:10). The immunoregulatoryeffect was confirmed with a significant decrease in the percentage ofIFN-γ⁺ CD4⁺ CD25⁻ T cells when pHSCs were added although less evident ascompared to the autoimmune assay, but still PD-L1 dependent (data notshown). This strongly confirms that pHSCs are endowed withPD-L1-dependent regulatory properties ex vivo.

Pharmacologically modulated HSCs reverted hyperglycemia—In order toevaluate the immunoregulatory properties in vivo of pHSCs, newlyhyperglycemic NOD mice were adoptively transferred with 3×10⁶ pHSCs.Infused pHSCs successfully reverted diabetes in 40% of NOD mice with 30%of treated hyperglycemic NOD mice remaining normoglycemic till thecompletion of the study at day 40. Kaplan-Meier curve showed a strongereffect of PD-L1⁺.Tg HSCs in reverting hyperglycemia in NOD mice, withpHSCs performing a little bit less better. The immuno-histopathologyanalysis of the pancreas of pHSC-treated hyperglycemic NOD mice revealedno evidence of infiltration of the islets or mild lymphocyteinfiltration with preserved insulin staining and reduced insulitis scoreas compared to untreated hyperglycemic NOD mice. Immunophenotype oftreated NOD-mice showed at day 40 after treatment a reduction in thepercentage of IFN-γ⁺ CD4⁺ and IL-17 and IFN-γ⁺ CD8⁺ T cells (data notshown). while no effect on Tregs (FoxP3⁺ regulatory CD4⁺ T cells) wasdetected. Quantification of IFN-γ-producing cells in an ex-vivo assay ofsplenocytes challenged with islets peptides at day 40 (BDC2.5, IGRP,GAD-65 and insulin) revealed a reduction of IFN-γ⁺ cells pHSC-treatedhyperglycemic NOD mice (data not shown).

PD-L1 defect is evident in human HSCs from T1D individuals—To assesswhether individuals with T1D displayed immunoregulatory defects inhematopoietic stem cells similar to the preclinical model, PD-L1expression was analyzed on HSCs extracted from the peripheral blood ofindividuals with T1D and healthy controls. In line with our findings inNOD mice, fewer PD-L1⁺ CD34⁺ cells were detectable in T1D individuals ascompared to healthy subjects (T1D=9.5% vs. controls=23.5%; p<0.001),(FIGS. 10A-10C). A western blot analysis and PCR analysis performed onRNA extracted from CD34⁺ cells previously isolated from PBMCs, confirmedPD-L1 reduced expression in HSCs obtained from healthy subjects ascompared to those obtained from T1D individuals (FIGS. 10D-10F).However, other immune relevant cells (e.g.; CD19⁺ B lymphocytes cells,CD11c⁺ dendritic cells and CD16⁺ cells) were not defective in PD-L1(data not shown), thus confirming that PD-L1 defect was mainlyrestrained to the hematopoietic stem cell populations in T1Dindividuals. We look at a confocal imaging of HSCs, by determining themerging of PD-L1 and CD34, in their primary site and niche (bone marrow)and determined that a PD-L1 defect is evident at their own niche as well(data not shown). We further looked at the frequencies of othercostimulatory molecules (e.g.; PD-L2 and PD-1) on peripheral HSCs fromT1D individuals and healthy controls, which did not appear to be reducedin T1D individuals. (data not shown). Next, we wanted to determine theeffect of high glucose on PD-L1 expression on HSCs. PBMCs were isolatedfrom peripheral blood of T1D individuals and CD34⁺ cells were sorted bymagnetic beads. CD34⁺ cells (HSCs) were cultured for 3 days in differentconditions (normal glucose, 20 mM high glucose and 35 mM high glucose).While some changes were evident in line with our findings in NOD mice,no particular pattern suggested the existence of any potential highglucose-associated effect on PD-L1 expression (data not shown). Then, weperformed a proliferation assay on CFSE labelled-HSCs from T1Dindividuals and controls, when cultured for 24 and 72 hours in SFEMIImedia. This was aimed to assess any apoptotic or survival-bias in ourexpression analysis. Our data indicated no difference in theproliferation rate of HSCs from T1D individuals and controls (data notshown). We have further studied HSC apoptotic rate. Although atbaseline, HSCs from T1D displayed a significantly higher percentage ofAnnexinV+/7-AAD− apoptotic cells as compared with HSCs from HC, this wasnot evident after 24 and 72 hours of culture, as both HSCs from T1D andHC individuals displayed a similar apoptotic rate (data not shown). Toconfirm that the mobilization of HSCs cells (CD34⁺) is not a therapeuticoption in the absence of a clear restoration of PD-L1 expression, weevaluated the mobilization properties of HSCs in T1D individuals. Wethus analyzed PD-L1 expression in a clinical trial (NCT01102699) inwhich 6 T1D individuals underwent HSCs mobilization with hrG-CSF (5μg/kg). While CD34⁺ cells significantly increased in healthy controls,an impaired mobilization of CD34⁺ was observed in T1D individuals. Thisdata confirms the existence of a HSC “mobilopathy” in T1D individuals.HSC immunophenotyping before and after mobilization with anti-CXCR4(Plerixafor) in 5 controls and 8 T1D individuals. The percentage ofCD34⁺ PD-L1⁺ cells decrease after in both T1D and controls, highlightingthat CD34⁺ cells require an in vitro manipulation to overturn PD-L1defect and recover their immunoregulatory properties (data not shown).

Pharmacologically modulated HSCs abrogate autoimmune response invitro—To overcome PD-L1 deficiency in human HSCs, we tested the effectof the same cocktail of small molecules that we developed in NOD mice.First, we evaluated PD-L1 expression in HSCs isolated from T1Dindividuals prior and post-modulation with a cocktail of smallmolecules. The newly human programmed HSCs (pHSCs) displayed anupregulation of PD-L1 expression as compared to unmodulated-HSCs.Immunofluorescence nicely depicted the increased surface PD-L1expression after modulation with small molecules, a combination ofgrowth factors (SCF, TPO, IL-3, IL-6, IFN-B, IFN-g and poly I:C) (datanot shown). Genome wide analysis of the pHSCs confirmed the upregulationof PD-L1 with nearly a 26-fold increase compared to the unmodulated HSCs(data not shown). To study whether HSCs cells or pHSCs isolated fromindividuals with T1D possess immunoregulatory functions ex vivo, PBMCsdepleted of HSCs were cocultured with HSCs or hpHSCs at 3 differentratios to PBMCs (1:1; 1:5 and 1:10) in the presence ofinsulin-associated autoantigen-2 (I-A2), and IFN-γ production by I-A2stimulated PBMCs was assessed in an ELISPOT assay. Interestingly,compared with PBMCs-IA-2-stimulated, the addition of HSCs resulted insignificantly (P<0.05) decrease of IFN-γ production. The suppression wasmore pronounced when hpHSCs were added (data not shown), suggesting thatHSCs and pHSCs are endowed with immunoregulatory activity. To furtherconfirm that the main immunosuppressive effect exerted by HSCs wasmainly due to PD-L1, we performed another Elispot assay to assess IFN-γproduction by PBMCs stimulated with IA-2 peptide, and pHSCs in thepresence of anti-PD-L1 blocking Ab or control Ab. Ab-mediated PD-L-1blockage hampered the immunoregulatory effect already exerted by pHSCsas revealed by the absence of an evident reduction in the percentage ofIFN-γ⁺ PBMCs (data not shown). We then tested the immunoregulatoryeffects of pHSCs in a non specific anti-CD3/CD28 assay. CD4⁺ T cellsextracted from HC individuals and stimulated by solubleanti-CD3/anti-CD28 were cocultured with HSCs or with pHSCs at 3different ratios to CD4⁺ T cells (1:1; 1:5 and 1:10). An evident andsignificant decrease in the percentage of IFN-γ⁺ CD4⁺ T cells wasremarkably observed when pHSCs were added (data not shown). The additionof anti-PD-L1 blocking Ab clearly abrogated the immunosuppressive effectof pHSCs mainly conferred by PD-L1 (data not shown). This stronglyconfirms that HSCs and pHSCs are endowed with PD-L1-dependent regulatoryproperties ex vivo. In order to evaluate the immunoregulatory propertiesin vivo of the newly generated pHSCs, NRG-Akita hyperglycemic mice havefirstly received human PBMCs (˜10×10⁶ cells) followed by islettransplantation with human islets (˜2000IEQ) and were then adoptivelytransferred with 1×10⁶ pHSCs (data not shown). Infused pHSCssuccessfully maintained NRG-Akita mice normoglycemic in NRG-Akita micetill the completion of the study. Kaplan-Meier curve showing reversal ofglycemia in different treated groups (data not shown). Theimmuno-histopathology analysis of the pancreas of treated mice withpHSCs revealed no evidence of infiltration of the islets or mildlymphocyte infiltration (data not shown) with preserved insulin stainingas compared to hyperglycemic untreated NOD and a reduced insulitisscore.

The references cited herein and throughout the specification areincorporated herein by reference.

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1-55. (canceled)
 56. A pharmaceutical composition comprising apopulation of modified, PD-L1+ expressing hematopoietic stem cells(HSCs), produced by an ex vivo method comprising: a) contacting a sampleof HSCs with a vector carrying an exogenous copy of a nucleic acidencoding a PD-L1 to modify the HSCs whereby the exogenous copy of anucleic acid is introduced into the HSCs; b) ex vivo culturing theresultant modified cells from the contacting; and c) establishing theexpression of PD-L1 on the modified HSCs, thereby producing thepopulation of modified HSCs cells expressing PD-L1.
 57. Thepharmaceutical composition of claim 56, wherein the ex vivo methodfurther comprises establishing that there is at least one-fold increasein the number of PD-L1+ expressing cells compared to non-modified cells.58. The pharmaceutical composition of claim 56, wherein the sample ofHSC is obtained from the bone marrow, umbilical cord, amniotic fluid,chorionic villi, cord blood, placental blood or peripheral blood. 59.The pharmaceutical composition of claim 56, wherein the sample of HSC isobtained from mobilized peripheral blood.
 60. The pharmaceuticalcomposition of claim 56, wherein the sample of HSCs is obtained from ahealthy individual.
 61. The pharmaceutical composition of claim 56,wherein the sample of HSCs is obtained from an individual with adiagnosed disease or disorder.
 62. The pharmaceutical composition ofclaim 56, wherein the diagnosed disease or disorder is an autoimmunedisease or disorder.
 63. The pharmaceutical composition of claim 56,wherein the autoimmune disease or disorder is Type 1 diabetes (T1D). 64.The pharmaceutical composition of claim 56, wherein the vector is viralvector.
 65. The pharmaceutical composition of claim 64, wherein theviral vector is a lentiviral vector.
 66. The pharmaceutical compositionof claim 56, wherein the nucleic acid is a complementary DNA (cDNA). 67.The pharmaceutical composition of claim 56, wherein the nucleic acid isa genomic DNA.
 68. The pharmaceutical composition of claim 56, whereinthe nucleic acid is integrated into the genome of the modified cells.